Semi-recombinant preparation of glp-1 analogues

ABSTRACT

A semi-recombinant method for the production of GLP-1 analogues and derivatives with non-proteogenic amino acids in the N-terminal part combining the use of recombinant expression techniques and chemical peptide synthesis.

FIELD OF THE INVENTION

The present invention is related to a semi-recombinant method for thepreparation of GLP-1 analogues and derivatives containingnon-proteogenic amino acids in the N-terminal part combining the use ofrecombinant expression techniques and chemical peptide synthesis.

BACKGROUND OF THE INVENTION

Human glucagon-like peptide-1 (GLP-1) is a 37 amino acid residuegastrointestinal hormone involved in the regulation of blood glucosemetabolism, gastrointestinal secretion and metabolism and food intake.GLP-1 is originating from preproglucagon which is synthesized inter aliain the L-cells in the distal ileum, in the pancreas and in the brain.GLP-1 stimulates insulin secretion in a glucose-dependant manner,stimulates insulin biosynthesis, promotes beta cell rescue, decreasesglucagon secretion, gastric emptying and food intake. Human GLP-1 ishydrolysed to GLP-1 (7-37) and GLP-1 (7-36)-amide which are bothinsulinotropic agents. Pharmacological doses of GLP-1 administered totype 2 diabetic patients have been shown to significantly raisecirculating insulin levels and to lower plasma glucagon levels. Theactions of GLP-1 are mediated by GLP-1 receptors in the pancreas, heart,kidney, central nervous system and gastrointestinal tract. Hence, GLP-1is expected to become very important in the treatment of diabetes.

However, native human GLP-1 is rapidly inactivated by degradation by theplasma enzyme dipeptidyl peptidase IV (DPP-IV) to a truncatedGLP-1(9-36)amide metabolite, which serves as a GLP-1 receptorantagonist. This confers the peptide with a short circulating half-life.

This short circulating half-life is a problem for many diabetes patientsparticularly in the type 2 diabetes segment who are subject to so-called“needle-phobia”, i.e. a substantial fear of injecting themselves. In thetype 2 diabetes segment most patients are treated with oralhypoglycaemic agents. Since GLP-1 compounds are expected to be aninjectable pharmaceutical product, the fear of injections may become aserious obstacle for the widespread use of these clinically verypromising compounds.

Hence, a range of different approaches and methods have been used formodifying the structure of GLP-1 compounds in order to provide a longerduration of action in vivo. Thus, a considerable effort is e.g. beingmade to develop analogues and derivatives of GLP-1 compounds lesssusceptible to DPP-IV mediated hydrolysis in order to reduce the rate ofdegradation by DPP-IV. WO 2006/097538, WO 2006/097536, WO 2006/037810,WO2006/005667, WO2005/058958, WO 2005/027978, WO 98/08871 and US2001/0011071 describe various GLP-1 analogues and derivatives, includingGLP analogues comprising non-proteogenic amino acids (i.e. non-naturalamino acids) which may confer a certain protection against hydrolysis byDPP-IV.

Polypeptides containing only proteogenic amino acids (i.e. natural aminoacids) such as native GLP-1, can be produced using recombinanttechniques or via chemical synthesis. However, polypeptides alsocontaining non-proteogenic amino acids such as N-terminally extendedGLP-1 analogues, cannot currently be prepared via recombinant expressiontechniques in a practical way and are in general prepared via chemicalsynthesis. The most widely used method for peptide synthesis is solidphase peptide synthesis where the adequate protected amino acids areincorporated in a stepwise manner using a polymer as a solid support.

Solid phase peptide synthesis (SPPS) can be very efficient in thepreparation of some peptides, but the use of protected amino acids incombination with the consistent use of excess of reactants makes thisapproach relatively expensive. In addition, each amino acid prolongationin a solid phase polypeptide synthesis requires a thorough washingprocedure. Typically, incorporation of one amino acid involves up to 10washings steps with solvents like NMP, DMF or DCM.

When polypeptides, such as insulinotropic agents, GLP-1 analogues,truncated analogues of GLP-1 and derivatives of GLP-1 are synthesizedusing SPPS, the formation of secondary structures during the synthesisoften leads to lower efficiency of the individual synthetic steps. As aconsequence, larger peptides or peptides containing certain amino acidsequences are often produced in low purity and yields. The impuritiesare often deletion peptides where one or more amino acids are missing inthe final sequence. These impurities can be very difficult to separatefrom the desired peptide, and result in a product contaminated withdeletion peptides.

It is the aim of the present invention to provide an efficient andeconomic method for the preparation of GLP-1 analogues and derivativeswhich are DPP-IV protected by having non-proteogenic amino acids in theN-terminal part. The method combines the advantage of cost-efficientproduction of truncated GLP-1 precursor molecules, using recombinanttechniques together with chemical synthesis of N-terminal extensionscomprising non-proteogenic amino acids. A significant reduction of thecost of producing GLP-1 analogues or derivatives is achieved. Lessexpensive GLP-1 analogues and derivatives are highly desirable formaximizing the number of patients for whom treatment is available, aswell as for exploiting the advantages of alternative delivery routeswhich have lower bioavailability than subcutaneous injection, e.g.transdermal and pulmonal delivery.

SUMMARY OF THE INVENTION

In its broadest aspect the present invention is related to a method forproducing GLP-1 analogues and derivatives being DPP-IV protected bycomprising one or more non-proteogenic amino acids in the N-terminalpart.

Thus, in one aspect the present invention is related to a method formaking GLP-1 analogues and derivatives comprising one or morenon-proteogenic amino acids, said method comprising the following steps:

(i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue or derivative under suitableconditions for expression of said precursor molecule,(ii) separating the expressed precursor molecule from the culture broth,(iii) coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the expressed precursor molecule,(iv) isolating the resulting GLP-1 analogue or derivative by suitablemeans, as known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the yeast plasmid pSA273. The plasmid contains anexpression cassette comprising an EcoRI-XbaI fragment inserted into theplasmid between the transcription-promoter and thetranscription-terminator of the S. cerevisiae TPI gene. The expressioncassette encodes a fusion product composed of the MFα1* pre-pro leader,a Lys-Arg cleavage site for the dibasic processing endopeptidase Kex2,and the GLP-1 precursor (R34)GLP-1(11-37).

FIG. 2 shows the NcoI-XbaI DNA fragments encoding the GLP-1 precursors.The GLP-1 precursor amino acid sequence encoded by the DNA fragments areindicated in bold. a) (R34)GLP-1(11-37); b)Ext1-(E22,R26,R34)GLP-1(8-37)K38-Ext2 and c)Ext1-(E22,R26,R34,K37)GLP-1(8-37)-Ext2; d)(R34)GLP-1(9-37).

FIG. 3 shows the NcoI-XbaI DNA fragments encoding the GLP-1 precursorsand the optimized leader sequences. (34R)GLP-1(11-37) is shown as anunlimiting example. Capital letters indicate the different sequences.Optimized amino acid sequences encoded by the DNA fragments areindicated in bold. ‘KR’ indicates a construct with the minimal Kex2pcleavage site LysArg.

FIG. 4 shows (34R)GLP-1(11-37) leader optimization as an example.Optimization of P1 to P6: (A-F). Incorporation of proline: (H, I).Charged amino acid incorporation into P7 to P11: (M-Z, K5-7, L2-4).Constructs for downstream processing with DAPase: (G, J, K, L). Yieldsare given in percent relative to a (34R)GLP-1(11-37) with theMFalpha*-leader and a simple LysArg Kex2p cleavage site.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed an economic and efficient methodfor preparing GLP-1 analogues and derivatives comprising one or morenon-proteogenic amino acids in the N-terminal part. Thus, it has beenfound that recombinantly produced GLP-1 precursor molecules whichcontain only proteogenic amino acids can be efficiently extended in theN-terminus using a non-proteogenic amino acid or a peptide containingone or more non-proteogenic amino acids, e.g. by reacting the precursorof the GLP-1 analogue with a carboxylic acid derivative, which isoptionally activated, of the extension fragment.

More specifically, the present invention provides a semi-recombinantmethod for preparation of GLP-1 analogues and derivatives containing oneor more non-proteogenic amino acids in the N-terminus, including thepositions 7, 8, 9 and/or 10. In addition, a useful method forderivatization of the ε-N of a lysine residue in the GLP-1 analogueprecursor molecule via acylation is provided. Hence, the presentinvention combines the cost efficient production of GLP-1 analogues andderivatives using recombinant techniques with the flexibility ofchemical synthesis.

In the present specification, the following terms have the indicatedmeaning:

The term “polypeptide” and “peptide” as used herein means a compoundcomposed of at least two constituent amino acids connected by peptidebonds. The constituent amino acids may be from the group of the aminoacids encoded by the genetic code and they may be natural amino acidswhich are not encoded by the genetic code, as well as synthetic aminoacids. Natural amino acids which are not encoded by the genetic code aree.g., γ-carboxyglutamate, ornithine, phosphoserine, D-alanine andD-glutamine. Synthetic amino acids comprise amino acids manufactured bychemical synthesis, i.e. D-isomers of the amino acids encoded by thegenetic code such as D-alanine and D-leucine, Aib (α-aminoisobutyricacid), Abu (α-aminobutyric acid), Tle (tert-butylglycine), β-alanine,3-aminomethyl benzoic acid, anthranilic acid.

The 22 proteogenic amino acids are: Alanine, Arginine, Asparagine,Aspartic acid, Cysteine, Cystine, Glutamine, Glutamic acid, Glycine,Histidine, Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine,Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine.

Thus, a non-proteogenic amino acid is a moiety which can be incorporatedinto a peptide via peptide bonds but is not a proteogenic amino acid.Examples of non-proteogenic aminoacids are, but not limited to,γ-carboxyglutamate, ornithine, phosphoserine, the D-amino acids such asD-alanine and D-glutamine. Synthetic non-proteogenic amino acidscomprise amino acids manufactured by chemical synthesis, i.e. D-isomersof the amino acids encoded by the genetic code such as D-alanine andD-leucine, Aib (α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tle(tert-butylglycine), 3-aminomethyl benzoic acid, anthranilic acid,des-amino-Histidine, the beta analogs of amino acids such as β-alanineetc., D-histidine, desamino-histidine, 2-amino-histidine,β-hydroxy-histidine, homohistidine, N^(α)-acetyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl) carboxylicacid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl) carboxylic acid,(1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylicacid, α-methyl prolin, 1-methyl histidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid3-(1,2,4-triazol-1-yl)-alanin.

The term “DPP-IV protected” as used herein referring to a polypeptidemeans a polypeptide which has been chemically modified in order torender said compound resistant to the plasma peptidase dipeptidylpeptidase-4 (DPP-IV). The DPP-IV enzyme in plasma is known to beinvolved in the degradation of several peptide hormones, e.g. GLP-1,GLP-2, Exendin-4 etc. Thus, a considerable effort is being made todevelop analogues and derivatives of the polypeptides susceptible tohydrolysis by DPP-IV in order to reduce the rate of degradation byDPP-IV. In one embodiment a DPP-IV protected peptide is more resistantto DPP-IV than GLP-1(7-37).

Resistance of a peptide to degradation by DPP-IV is determined by thefollowing degradation assay:

Aliquots of the peptide (5 nmol) are incubated at 37° C. with 1 μL ofpurified DPP-IV corresponding to an enzymatic activity of 5 mU for10-180 minutes in 100 μL of 0.1 M triethylamine-HCl buffer, pH 7.4.Enzymatic reactions are terminated by the addition of 5 μL of 10%trifluoroacetic acid, and the peptide degradation products are separatedand quantified using HPLC analysis. One method for performing thisanalysis is: The mixtures are applied onto a Vydac C18 widepore (30 nmpores, 5 μm particles) 250×4.6 mm column and eluted at a flow rate of 1ml/min with linear stepwise gradients of acetonitrile in 0.1%trifluoroacetic acid (0% acetonitrile for 3 min, 0-24% acetonitrile for17 min, 24-48% acetonitrile for 1 min) according to Siegel et al.,Regul. Pept. 1999; 79:93-102 and Mentlein et al. Eur. J. Biochem. 1993;214:829-35. Peptides and their degradation products may be monitored bytheir absorbance at 220 nm (peptide bonds) or 280 nm (aromatic aminoacids), and are quantified by integration of their peak areas related tothose of standards. The rate of hydrolysis of a peptide by dipeptidylpeptidase DPP-IV is estimated at incubation times which result in lessthan 10% of the peptide being hydrolysed.

In its broadest sense, the term “GLP-1 analogue” as used herein means ananalogue of a molecule of the glucagon family of peptides or an analogueof the family of exendins. The glucagon family of peptides are encodedby the pre-proglucagon gene and encompasses three small peptides with ahigh degree of homology, i.e. glucagon (1-29), GLP-1 (1-37) and GLP-2(1-33). Exendins are peptides expressed in lizards and like GLP-1, areinsulinotropic. Examples of exendins are exendin-3 and exendin-4.

The term “analogue” as used herein referring to a polypeptide means amodified peptide wherein one or more amino acid residues of the peptidehave been substituted by other amino acid residues and/or wherein one ormore amino acid residues have been deleted from the peptide and/orwherein one or more amino acid residues have been added to the peptide.Such addition or deletion of amino acid residues can take place at theN-terminal of the peptide and/or at the C-terminal of the peptide. Thusa GLP-1 analogue according to the invention is an analogue of GLP-1(1-37) according to the invention wherein one or more amino acidresidues of the peptide have been substituted by other amino acidresidues and/or wherein one or more amino acid residues have beendeleted from the peptide and/or wherein one or more amino acid residueshave been added to the peptide.

For the present purposes any amino acid substitution, deletion, and/oraddition refers to the sequence of human GLP-1(7-37) which is includedherein as SEQ ID NO: 1. However, the numbering of the amino acidresidues in the sequence listing always starts with no. 1, whereas forthe present purpose we want, following the established practice in theart, to start with amino acid residue no. 7 and assign number 7 to it.Therefore, generally, any reference herein to a position number of theGLP-1(7-37) sequence is to the sequence starting with His at position 7and ending with Gly at position 37.

A simple system is often used to describe GLP-1 analogues: for example[Arg³⁴]GLP-1(7-37)Lys designates a GLP-1(7-37) analogue wherein thenaturally occurring lysine at position 34 has been substituted witharginine and wherein a lysine has been added to the C-terminal. Anotherexample [Arg³⁴]GLP-1(11-37) designates a GLP-1 analogue wherein thenaturally occurring lysine at position 34 has been substituted witharginine and the amino acids in positions 7, 8, 9 and 10 are absent.

The expression “a position equivalent to” when used herein tocharacterize a modified GLP-1(7-37) sequence refers to the correspondingposition in the natural GLP-1(7-37) sequence (having the sequence of SEQID NO: 1). Corresponding positions are easily deduced, e.g. by simplehandwriting and eyeballing. In the alternative, a standard protein orpeptide alignment program may be used, such as “align” which is aNeedleman-Wunsch alignment. The algorithm is described in Needleman, S.B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48: 443-453,and the align program by Myers and W. Miller in “Optimal Alignments inLinear Space” CABIOS (computer applications in the biosciences) (1988)4:11-17. For the alignment, the default scoring matrix BLOSUM50 and thedefault identity matrix may be used, and the penalty for the firstresidue in a gap may be set at −12 and the penalties for additionalresidues in a gap at −2.

The term “derivative” as used herein in relation to a peptide means achemically modified peptide or an analogue thereof, wherein at least onesubstituent has been attached to the unmodified peptide or an analoguethereof, i.e. a peptide which has been covalently modified. Typicalmodifications are amides, carbohydrates, alkyl groups, acyl groups,esters and the like. An example of a derivative of GLP-1(7-37) isN^(ε26)-(γ-Glu(N^(α)-hexadecanoyl)))-[Arg³⁴, Lys²⁶])GLP-1(7-37).

All amino acids for which the optical isomer is not stated is to beunderstood to mean the L-isomer.

In embodiments of the invention a maximum of 17 amino acids in the GLP-1analogue have been modified (substituted, deleted, added or anycombination thereof) relative to human GLP-1(7-37). In embodiments ofthe invention a maximum of 15 amino acids in the GLP-1 analogue havebeen modified. In embodiments of the invention a maximum of 10 aminoacids in the GLP-1 analogue have been modified. In embodiments of theinvention a maximum of 8 amino acids in the GLP-1 analogue have beenmodified. In embodiments of the invention a maximum of 7 amino acids inthe GLP-1 analogue have been modified. In embodiments of the invention amaximum of 6 amino acids in the GLP-1 analogue have been modified. Inembodiments of the invention a maximum of 5 amino acids in the GLP-1analogue have been modified. In embodiments of the invention a maximumof 4 amino acids in the GLP-1 analogue have been modified. Inembodiments of the invention a maximum of 3 amino acids in the GLP-1analogue have been modified. In embodiments of the invention a maximumof 2 amino acids in the GLP-1 analogue have been modified. Inembodiments of the invention 1 amino acid in the GLP-1 analogue has beenmodified.

In one embodiment the GLP-1 analogue is an insulinotropic agent.

In one embodiment the GLP-1 analogue is a GLP-1 agonist.

The term “GLP-1 agonist” as used herein refers to any glucagon-likepeptide which fully or partially activates the human GLP-1 receptor. Ina preferred embodiment, the “GLP-1 agonist” is any glucagon-like peptidethat binds to a GLP-1 receptor, preferably with an affinity constant(KD) or a potency (EC₅₀) of below 1 μM, e.g., below 100 nM as measuredby methods known in the art (see e.g., WO 98/08871) and exhibitsinsulinotropic activity, where insulinotropic activity may be measuredin vivo or in vitro assays known to those of ordinary skill in the art.For example, the GLP-1 agonist may be administered to an animal and theinsulin concentration measured over time.

In one aspect of the invention, the GLP-1 analogue to be included in thepharmaceutical compositions of the present invention is an analogue ofGLP-1, wherein the analogue comprises at least one non-proteogenicpeptide.

In one aspect of the invention, the GLP-1 analogue is selected from thegroup consisting of Aib^(8,22,35) GLP-1(7-37), Aib^(8,35) GLP-1(7-37),Aib^(8,22) GLP-1(7-37), Aib^(8,22,35) Arg^(26,34)Lys³⁸GLP-1(7-38),Aib^(8,35) Arg^(26,34)Lys³⁸GLP-1(7-38), Aib^(8,22)Arg^(26,34)Lys³⁸GLP-1(7-38), Aib^(8,22,35) Arg^(26,34)Lys³⁸GLP-1(7-38),Aib^(8,35) Arg^(26,34)Lys³⁸GLP-1(7-38), Aib^(8,22,35)Arg²⁶Lys³⁸GLP-1(7-38), Aib^(8,35) Arg²⁶Lys³⁸GLP-1(7-38), Aib^(8,22)Arg²⁶Lys³⁸GLP-1(7-38), Aib^(8,22,35) Arg³⁴Lys³⁸GLP-1(7-38),Aib^(8,35)Arg³⁴Lys³⁸GLP-1(7-38), Aib^(8,22)Arg³⁴Lys³⁸GLP-1(7-38),Aib^(8,22,35)Ala³⁷Lys³⁸GLP-1(7-38), Aib^(8,35)Ala³⁷Lys³⁸GLP-1(7-38),Aib^(8′22)Ala³⁷Lys³⁸GLP-1(7-38), Aib^(8,22,35) Lys³⁷GLP-1(7-37),Aib^(8,35)Lys³⁷GLP-1(7-37) and Aib^(8,22)Lys³⁷GLP-1(7-38).

In one aspect of the invention, the GLP-1 analogue is selected from thegroup consisting of

-   [desaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1(7-37)amide,-   [desaminoHis⁷,Arg³⁴]GLP-1-(7-37),    [Aib⁸,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷, Glu²² Arg²⁶, Arg³⁴, Phe(m-CF3)²⁸]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴]GLP-1-(7-37)-Lys,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴]GLP-1-(7-37)-Lys,-   [desaminoHis⁷,Arg²⁶,Arg³⁴,]GLP-1-(7-37)-Lys,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [DesaminoHis⁷,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [DesaminoHis⁷,Glu²²,Arg²⁶,Glu³⁰,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [Aib⁸,Lys²⁰,Arg²⁶,Glu³⁰,Thr(O-benzyl)³³,]GLP-1-(7-37)amide,-   [Aib⁸,Glu²²,Arg²⁶,Lys³⁰]GLP-1-(7-37), [Aib⁸, Glu²²,    Arg²⁶,Lys³¹]GLP-1-(7-37),-   [Aib⁸,Lys²⁰,Arg²⁶,2-Naphtylalanine²⁸, Glu³⁰,]GLP-1 (7-37)amide,-   [Aib⁸, Glu²², Arg²⁶, Arg³⁴,]GLP-1-(7-37)-Lys,-   [Aib⁸,Lys²⁰,Arg²⁶, 2-Naphtylalanine12, Glu³⁰,]GLP-1-(7-37)amide,-   [Aib⁸,Glu²²,Arg²⁶,Lys³¹,Arg³⁴]GLP-1-(7-37),-   [Aib⁸,Arg³⁴]GLP-1-(7-37),-   [Aib⁸,Arg³⁴]GLP-1-(7-37)-amide,-   [Aib⁸,Lys¹⁸,Arg²⁶,Arg³⁴]GLP-1(7-37),-   [Aib⁸,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [Aib⁸, Lys²⁶]GLP-1 (7-37)amide,-   [Aib⁸,Arg³⁴]GLP-1-(7-34),-   [Aib⁸,Arg³⁴]GLP-1-(7-35),-   [Aib⁸,Lys³³,Arg³⁴]GLP-1-(7-34),-   [Aib⁸,Arg³⁴]GLP-1-(7-36)amide,-   [Aib⁸,Lys²⁶,Arg³⁴]GLP-1-(7-36)amide,-   [Aib⁸,Glu²²,Arg²⁶,Arg³⁴]GLP-1-(7-37)Lys,-   [Aib⁸,Lys²⁰,Glu²²,Arg²⁶,Glu³⁰,Pro³⁷]GLP-1-(7-37)amide,-   [Aib⁸,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Arg26,Arg34,Lys^(37])GLP-1-(7-37)amide,-   Aib⁸,Glu²²,Arg²⁶,Glu³⁰,Pro³⁷NLP-1-(7-37)Lys,-   [Aib⁸,Glu²²,Arg²⁶,Glu³⁰,Pro³⁷]GLP-1-((7-37)Lys,-   [Aib⁸,Arg²⁶,Arg³⁴]GLP-1-(7-37).

In yet another embodiment, the GLP-1 analogue to be included in thepharmaceutical compositions of the present invention is an analogue ofGLP-2, wherein the analogue comprises at least one non-proteogenicpeptide.

In yet another embodiment the GLP-1 analogue is an analogue of exendin-4or exendin-3, wherein the analogue comprises at least onenon-proteogenic peptide. Examples of exendins as well as analoguesthereof are disclosed in WO 97/46584, U.S. Pat. No. 5,424,286 and WO01/04156. U.S. Pat. No. 5,424,286 describe a method for stimulatinginsulin release with an exendin peptide. The exendin peptides disclosedinclude HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX (SEQ ID NO. 12), wherein X-P orY, and wherein exendin-3 is HSDGTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS(SEQ ID NO. 13) and exendin-4 (1-39) isHGEGTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO. 2). WO 97/46584describes truncated versions of exendin peptide(s). The disclosedpeptides increase secretion and biosynthesis of insulin, but reducethose of glucagon. WO 01/04156 describes exendin-4 analogues andderivatives as well as the preparation of these molecules.

The term “exendin-4 peptide” as used herein means exendin-4(1-39) (SEQID NO. 2), an exendin-4(1-39) analogue, an exendin-4(1-39) derivative ora derivative of an exendin-4(1-39) analogue, insulinotropic fragmentsthereof, insulinotropic analogs thereof and insulinotropic derivativesthereof. Insulinotropic fragments of exendin-4 are insulinotropic agentsfor which the entire sequence can be found in the sequence of exendin-4(SEQ ID NO. 2) and where at least one terminal amino acid has beendeleted. Examples of insulinotropic fragments of exendin-4(1-39) areexendin-4(1-38) and exendin-4(1-31).

The insulinotropic property of a compound may be determined by in vivoor in vitro assays well known in the art. For instance, the compound maybe administered to an animal and monitoring the insulin concentrationover time. Insulinotropic analogs of exendin-4(1-39) refer to therespective molecules wherein one or more of the amino acids residueshave been exchanged with other amino acid residues and/or from which oneor more amino acid residues have been deleted and/or from which one ormore amino acid residues have been added with the proviso that saidanalogue either is insulinotropic or is a prodrug of an insulinotropiccompound. An example of an insulinotropic analog of exendin-4(1-39) isSer²Asp³-exendin-4(1-39) wherein the amino acid residues in position 2and 3 have been replaced with serine and aspartic acid, respectively(this particular analog also being known in the art as exendin-3).Insulinotropic derivatives of exendin-4(1-39) and analogs thereof arewhat the person skilled in the art considers to be derivatives of thesepeptides, i.e., having at least one substituent which is not present inthe parent peptide molecule with the proviso that said derivative eitheris insulinotropic or is a prodrug of an insulinotropic compound.Examples of substituents are amides, carbohydrates, alkyl groups, estersand lipophilic substituents. An example of an insulinotropic derivativesof exendin-4(1-39) and analogs thereof is Tyr³¹-exendin-4(1-31)-amide.

The term “stable exendin-4 compound” as used herein means a chemicallymodified exendin-4(1-39), i.e., an acylated exendin-3 or acylatedexendin-4 analogue which exhibits an in vivo plasma eliminationhalf-life of at least 10 hours in man, as determined by conventionalmethods.

All amino acids for which the optical isomer is not stated is to beunderstood to mean the L-isomer.

The term “insulinotropic agent” as used herein means a compound which isan agonist of the human GLP-1 receptor, i.e. a compound which stimulatesthe formation of cAMP in a suitable medium containing the human GLP-1receptor (one such medium disclosed below). The potency of aninsulinotropic agent is determined by calculating the EC₅₀ value fromthe dose-response curve as described below.

Baby hamster kidney (BHK) cells expressing the cloned human GLP-1receptor (BHK-467-12A) were grown in DMEM media with the addition of 100IU/mL penicillin, 100 μg/mL streptomycin, 5% fetal calf serum and 0.5mg/mL Geneticin G-418 (Life Technologies). The cells were washed twicein phosphate buffered saline and harvested with Versene. Plasmamembranes were prepared from the cells by homogenisation with anUltraturrax in buffer 1 (20 mM HEPES-Na, 10 mM EDTA, pH 7.4). Thehomogenate was centrifuged at 48,000×g for 15 min at 4° C. The pelletwas suspended by homogenization in buffer 2 (20 mM HEPES-Na, 0.1 mMEDTA, pH 7.4), then centrifuged at 48,000×g for 15 min at 4° C. Thewashing procedure was repeated one more time. The final pellet wassuspended in buffer 2 and used immediately for assays or stored at −80°C.

The functional receptor assay was carried out by measuring cyclic AMP(cAMP) as a response to stimulation by the insulinotropic agent. cAMPformed was quantified by the AlphaScreen™ cAMP Kit (Perkin Elmer LifeSciences). Incubations were carried out in half-area 96-well microtiterplates in a total volume of 50 μL buffer 3 (50 mM Tris-HCl, 5 mM HEPES,10 mM MgCl₂, pH 7.4) and with the following additions: 1 mM ATP, 1 μMGTP, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.01% Tween-20, 0.1%BSA, 6 μg membrane preparation, 15 μg/mL acceptor beads, 20 μg/mL donorbeads preincubated with 6 nM biotinyl-cAMP. Compounds to be tested foragonist activity were dissolved and diluted in buffer 3. GTP was freshlyprepared for each experiment. The plate was incubated in the dark withslow agitation for three hours at room temperature followed by countingin the Fusion™ instrument (Perkin Elmer Life Sciences).Concentration-response curves were plotted for the individual compoundsand EC₅₀ values estimated using a four-parameter logistic model withPrism v. 4.0 (GraphPad, Carlsbad, Calif.).

The term “semi-recombinant” as used herein refers to a method comprisingthe steps of preparing a first peptide fragment such as a first fragmentof a GLP-1 analogue by a recombinant process, i.e. from a culture ofhost cells, and subsequently coupling a second fragment of a peptide tothe first fragment of the peptide, wherein the second fragment isprepared chemically, i.e. in solution or on a resin using solid phasepeptide chemistry. The coupling of the fragments may be performed eitherchemically or enzymatically.

The term “separating the expressed precursor molecule” as used hereinmeans any kind of separation, including isolation of the recombinantlyproduced compound and also includes disintegrating or permeabilising ahost cell.

Accordingly, the present invention relates to a method for making aGLP-1 analogue or derivative comprising one or more non-proteogenicamino acids in the N-terminal part, said method comprising the steps of:

(i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue or derivative under suitableconditions for expression of said precursor molecule,(ii) separating the expressed precursor molecule from the culture broth,(iii) coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the expressed precursor molecule,(iv) isolating the resulting GLP-1 analogue or derivative by suitablemeans, as known in the art.

In the present context, the term “precursor molecule of the GLP-1analogue”, is to be understood as a peptide derived from GLP-1, GLP-2,Exendin-3 or Exendin-4 peptides, i.e., a GLP-1 analogue sequence, beforeaddition of an N-terminal amino acid extension. Examples include, butare not limited to human GLP-1 (11-37) or an analogue thereof, humanGLP-1 (9-37) or an analogue thereof and human GLP-1 (8-37) or ananalogue thereof.

Accordingly, in one aspect the precursor molecule of the GLP-1 analogueaccording to the invention, comprises a GLP-1 peptide derived from theGLP-1 (7-37) sequence as shown in SEQ ID NO:1.

Exendin-4 is a 39 amino acid residue peptide isolated from the venom ofGila monster lizards (Heloderma suspectum and Heloderma horridum). Thispeptide shares 52% homology with GLP-1 (7-37) in the overlapping region.Exendin-4 is a potent GLP-1 receptor agonist and has also been shown tostimulate insulin release and ensuing lowering of the blood glucoselevel when injected into dogs.

Thus, due to the 52% homology with GLP-1 (7-37) in the overlappingregion, the precursor molecule of the GLP-1 analogue according to theinvention, may in certain embodiments be derived from an exendin-4peptide as shown in SEQ ID NO:2.

Accordingly, the precursor molecules of the GLP-1 analogue may in afurther aspect comprise an amino acid sequence of the general formula:

(SEQ ID NO: 3) Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅wherein

Xaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr or Gln;Xaa₂₀ is Leu or Met; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys or Arg;Xaa₂₅ is Ala or Val; Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu;Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu,Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆ is Arg, Gly or Lys;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or is absent;Xaa₃₈ is Lys, Ser, or is absent.Xaa₃₉ is Ser, Lys, or is absent;Xaa₄₀ is Gly, or is absent;Xaa₄₁ is Ala, or is absent;Xaa₄₂ is Pro, or is absent;Xaa₄₃ is Pro, or is absent;Xaa₄₄ is Pro, or is absent;Xaa₄₅ is Ser, or is absent;provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ orXaa₄₅ is absent then each amino acid residue downstream is also absent.

In a further aspect of the invention, the precursor molecule of theGLP-1 analogue is selected from the list of precursor moleculescomprising the amino acid sequence of the general formula:

(SEQ ID NO: 4) Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄- Xaa₄₅wherein

Xaa₉ is Glu or Asp Xaa₁₀ is Gly or Ala Xaa₁₆ is Val or Leu; Xaa₁₈ isSer, Lys or Arg; Xaa₁₉ is Tyr or Gln; Xaa₂₀ is Leu or Met; Xaa₂₂ is Glyor Glu; Xaa₂₃ is Gln, Glu, Lys or Arg; Xaa₂₅ is Ala or Val; Xaa₂₆ isLys, Glu or Arg; Xaa₂₇ is Glu or Leu; Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ isVal, Lys or Arg; Xaa₃₄ is Lys, Glu, Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆is Arg, Gly or Lys;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or is absent;Xaa₃₈ is Lys, Ser, or is absent.Xaa₃₉ is Ser, Lys, or is absent;Xaa₄₀ is Gly, or is absent;Xaa₄₁ is Ala, or is absent;Xaa₄₂ is Pro, or is absent;Xaa₄₃ is Pro, or is absent;Xaa₄₄ is Pro, or is absent;Xaa₄₅ is Ser, or is absent;provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ orXaa₄₅ is absent then each amino acid residue downstream is also absent.

In a further aspect of the invention, the precursor molecule of theGLP-1 analogue is selected from the precursor molecules comprising anamino acid sequence of the general formula:

(SEQ ID NO: 5) Xaa₈-Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃- Xaa₄₄-Xaa₄₅wherein

Xaa₈ is Ala, Gly, Val, Leu, Ile or Lys; Xaa₉ is Glu or Asp; Xaa₁₀ is Glyor Ala; Xaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr orGln; Xaa₂₀ is Leu or Met; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys orArg; Xaa₂₅ is Ala or Val; Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu;Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu,Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆ is Arg, Gly or Lys;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or is absent;Xaa₃₈ is Lys, Ser, or is absent;Xaa₃₉ is Ser, Lys, or is absent;Xaa₄₀ is Gly, or is absent;Xaa₄₁ is Ala, or is absent;Xaa₄₂ is Pro, or is absent;Xaa₄₃ is Pro, or is absent;Xaa₄₄ is Pro, or is absent;Xaa₄₅ is Ser, or is absent;provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ orXaa₄₅ is absent then each amino acid residue downstream is also absent.

The GLP-1 analogue precursor molecules of the present invention may beprepared by means of recombinant DNA technology using general methodsand principles known to the person skilled in the art, by culturing asuitable host cell comprising a nucleotide sequence encoding a precursormolecule of the GLP-1 analogue under suitable conditions for expressionof the precursor molecule.

The nucleic acid sequence encoding the precursor molecule of the GLP-1analogue may be prepared synthetically by established standard methodswell known in the art, e.g. by synthesizing oligonucleotides in anautomatic DNA synthesizer, and subsequently purify, anneal, ligate andclone the nucleic acid sequences into suitable vectors.

The cloning of the nucleic acid sequences encoding the precursormolecule of the GLP-1 analogue can be effected, e.g., by using the wellknown polymerase chain reaction (PCR).

The nucleic acid sequence encoding the precursor molecule of the GLP-1analogue is inserted into a recombinant expression vector which may beany vector which may conveniently be subjected to recombinant DNAprocedures.

The choice of vector will often depend on the host cell into which it isto be introduced. Thus, the vector may be an autonomously replicatingvector, i.e., a vector which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g., aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

For extracellular products the proteinaceous components of thesupernatant are isolated by filtration, column chromatography orprecipitation, e.g. microfiltration, ultrafiltration, isoelectricprecipitation, purification by a variety of chromatographic procedures,e.g. ion exchange chromatography, hydrophobic interactionchromatography, gel filtration chromatography, affinity chromatography,or the like, dependent on the type of polypeptide in question. Forintracellular or periplasmic products the cells isolated from theculture medium are disintegrated or permeabilised and extracted torecover the product polypeptide or precursor thereof.

The DNA sequence encoding the precursor molecule of the GLP-1 analoguemay suitably be of genomic or cDNA origin, for instance obtained bypreparing a genomic or cDNA library and screening for DNA sequencescoding for all or part of the peptide by hybridisation using syntheticoligonucleotide probes in accordance with standard techniques (see, forexample, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).The DNA sequence encoding the polypeptide may also be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by Beaucage and Caruthers, Tetrahedron Letters 22(1981), 1859-1869, or the method described by Matthes et al., EMBOJournal 3 (1984), 801-805. The DNA sequence may also be prepared bypolymerase chain reaction using specific primers, for instance asdescribed in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239(1988), 487-491.

The DNA sequence encoding the precursor molecule of the GLP-1 analoguemay be inserted into any vector which may conveniently be subjected torecombinant DNA procedures, and the choice of vector will often dependon the host cell into which it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome (s) intowhich it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the precursor molecule of the GLP-1 analogue is operably linkedto additional segments required for transcription of the DNA, such as apromoter. The promoter may be any DNA sequence which showstranscriptional activity in the host cell of choice and may be derivedfrom genes encoding proteins either homologous or heterologous to thehost cell. Examples of suitable promoters for directing thetranscription of the DNA encoding the peptide of the invention in avariety of host cells are well known in the art, cf. for instanceSambrook et al., supra.

The DNA sequence encoding the precursor molecule of the GLP-1 analoguemay also, if necessary, be operably connected to a suitable terminator,polyadenylation signals, transcriptional enhancer sequences, andtranslational enhancer sequences. The recombinant vector of theinvention may further comprise a DNA sequence enabling the vector toreplicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate. For large scalemanufacture the selectable marker preferably is not antibioticresistance, e.g. antibiotic resistance genes in the vector arepreferably excised when the vector is used for large scale manufacture.Methods for eliminating antibiotic resistance genes from vectors areknown in the art, see e.g. U.S. Pat. No. 6,358,705 which is incorporatedherein by reference.

To direct precursor molecule of the GLP-1 analogue of the presentinvention into the secretory pathway of the host cells, a secretorysignal sequence (also known as a leader sequence, prepro sequence or presequence) may be provided in the recombinant vector. The secretorysignal sequence is joined to the DNA sequence encoding the peptide inthe correct reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the peptide. The secretorysignal sequence may be that normally associated with the peptide or maybe from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the precursormolecule of the GLP-1 analogue, the promoter and optionally theterminator and/or secretory signal sequence, respectively, and to insertthem into suitable vectors containing the information necessary forreplication, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the precursormolecule of the GLP-1 analogue and includes, but is not limited to,mammalian host cells, avian host cells, insect host cells, plant hostcells, bacterial host cells, fungal host cells and yeast host cells.Examples of suitable host cells well known and used in the art include,but are not limited to Saccharomyces cerevisiae, Pichia pastoris andother yeasts, E. coli, Bacillus subtilis, and Pseudomonas fluorescens ormammalian BHK or CHO cell lines.

The methods of introducing exogenous nucleic acid into the hosts arewell known in the art, and will vary with the host cell used. The hostcell, when cultured under appropriate conditions, synthesizes a GLP-1precursor molecule which can subsequently be collected from the culturemedium (if the host cell secretes it into the medium) or directly fromthe host cell producing it (if it is not secreted).

In one embodiment, GLP-1 precursor molecules are produced in yeastcells. Yeast expression hosts are well known in the art, and include butare not limited to Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii, Pichia methanolica and Pichia pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica.

In a one embodiment, GLP-1 precursor molecules are produced in bacterialsystems. Bacterial expression hosts are well known in the art, andinclude but are not limited to E. coli, Bacillus subtilis, Pseudomonasfluorescens, Lactococcus lactis, Streptococcus cremoris, andStreptococcus lividans.

In one embodiment, GLP-1 precursor molecules are expressed infilamentous fungi. Filametous fungi hosts are well known in the art, andinclude but are not limited to Neurospora, Penicillium, Tolypocladium,and Aspergillus hosts such as Aspergillus nidulans, Aspergillus oryzeaand Aspergillus niger.

In one embodiment, GLP-1 precursor molecules are produced in insectcells. Expression systems have been developed for Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melangaster, Spodopterafrugiperda, and Trichoplusia ni. Each of these cell lines is known byand available to those skilled in the art of protein expression.

In one embodiment, GLP-1 precursor molecules are produced in otherhigher eukaryotic cells. These include but are not limited to aviancells and plant cells such as Chlamydomonas reinhardtii.

In one embodiment, GLP-1 precursor molecules are produced in mammalianhost cell lines. Examples of these include, but are not limited to Babyhamster kidney (BHK), Chinese hamster ovary (CHO) and COS cells. Morespecific examples include, but are not limited to monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293); human liver cells (Hep G2, HB 8065).

The appropriate host cell is selected by the person the skill in theart. The medium used to culture the host cells may be any conventionalmedium suitable for growing the host cells, such as minimal or complexmedia containing appropriate supplements. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedrecipes (e.g. in catalogues of the American Type Culture Collection).

The precursor molecule of the GLP-1 analogue produced by the cells maythen be separated from the culture broth or culture medium byconventional procedures including separating the host cells from themedium by centrifugation or filtration.

As mentioned above, it is an aspect of the present invention to couplean N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the expressed GLP-1 analogue precursormolecule, in order to render the compound more resistant to the plasmapeptidase dipeptidyl peptidase-4 (DPP-IV).

Such non-proteogenic amino acids may be selected fromγ-carboxyglutamate, ornithine, phosphoserine, D-amino acids such asD-alanine and D-glutamine, D-alanine and D-leucine, Aib(α-aminoisobutyric acid), Abu (α-aminobutyric acid), Tle(tert-butylglycine), 3-aminomethyl benzoic acid, anthranilic acid,des-amino-Histidine, β-alanine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,Nα-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine, 4-pyridylalanine,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid,(1-aminocyclooctyl) carboxylic acid, α-methyl prolin, 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine. The desamino-histidine, although notcontaining an amino group, is thus herein referred to as anon-proteogenic amino acid, for ease in classification and nomenclature.

In one aspect of the invention, the length of the N-terminal amino acidextension comprising one or more non-proteogenic amino acids is 1 aminoacid, 2 amino acids, 3 amino acids or 4 amino acids.

In one aspect of the invention, the length of the N-terminal amino acidextension comprising one or more non-proteogenic amino acids is 1 aminoacid, 2 amino acids or 4 amino acids.

In one aspect of the invention, the length of the N-terminal amino acidextension comprising one or more non-proteogenic amino acids is 1 aminoacid or 2 amino acids.

In one aspect of the invention, the length of the N-terminal amino acidextension comprising one or more non-proteogenic amino acids is 2 aminoacids.

In one aspect of the invention, the length of the N-terminal amino acidextension comprising one or more non-proteogenic amino acids is 1 aminoacid.

It is contemplated that not all of the amino acids of the N-terminalamino acid extension necessarily are non-proteogenic amino acids. Hence,in certain aspects the N-terminal amino acid extension comprises 1, 2, 3or 4 non-proteogenic amino acids.

In one aspect of the invention the N-terminal amino acid extensioncomprising one or more non-proteogenic amino acids has the generalformula

Xaa₇-Xaa₈-Xaa₉-Xaa₁₀

whereinXaa₇ is selected from L-histidine, D-histidine, desamino-histidine,2-amino-histidine, (β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine, 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine;Xaa₈ is selected from Ala, Gly, Val, Leu, Ile, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid and alpha-methyl prolineXaa₉ is selected from Glu, Asp, γ,γ-dimethyl Glu, β,β-dimethyl Glu andβ,β-dimethyl Asp, andXaa₁₀ is selected from Gly, Aib, (1-aminocyclopropyl) carboxylic acid,(1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylicacid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)carboxylic acid, and (1-aminocyclooctyl) carboxylic acid.

The free carboxylic acid of the sequence Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ mayefficiently be prepared in a solution or on a resin such as2-chlorotritylchloride resin e.g. via an Fmoc based strategy usingadequately protected building blocks using appropriate reactionconditions described in references (Organic Synthesis on solid Phase,Florencio Zaragoza Dorwald, Wiley-VCH Verlag GmbH, D-69469 Weinheim,2000), (Novabiochem Catalog, Merck Biosciences 2006/2007) and (FmocSolid Phase Peptide Synthesis, Edited by W. C. Chan and P. D. White,Oxford University Press, 2000, ISBN 0-19-963724-5). The amino acids inthe sequence can be protected or unprotected. The N-terminal extensionpeptide of sequence Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ may be activated to anactivated acylating agent. The term “activated” acylating agent means anacylating agent which has been activated using general techniques ase.g. described in “Amide bond formation and peptide coupling”[Tetrahedron 61(46), 10827-10852 (2005)]. Examples of activated estersinclude, but are not limited to acid chloride, acid bromides, acidfluorides, symmetrical anhydride, mixed anhydride, carboxylic acidsactivated using common carbodiimides such as but not limited todiisopropylcarbodiimide (DIPCDI), N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC).Furthermore, included, but not limited to, are carboxylic acids usingthe aforementioned carbodiimides and an additive such as but not limitedto N-hydroxybenzotriazol (HOBt), 1-Hydroxy-7-azabenzotriazole,6-chloro-N-hydroxybenzotriazol (HOAt),3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (DhbtOH). Also included,but not limited to, are carboxylic acids activated with an uronium saltor a phosphonium salt, such as but not limited toO-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU), 2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU),(2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumtetrafluoroborate) (TCTU),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU),1-benzotriazolyoxytris-(dimethylamino)phosphonium hexafluorophosphate(BOP) benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PYBOP). Other activated esters include, but are not limited to estersof N-hydroxysuccinimid (NHS ester), pentafluorophenol ester (PfP-ester),2,4-dinitrophenyl ester, 4-nitrophenyl ester,3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) andCarbonyldiimidazole (CDI), N-ethyl-5-phenylisoxazolium-3′-sulfonate(NEPIS), preferably a N-hydroxysuccinimide ester or a HOBt ester or aderivative hereof using reaction conditions described in references(Organic Synthesis on solid Phase, Florencio Zaragoza Dorwald, Wiley-VCHVerlag GmbH, D-69469 Weinheim, 2000), (Novabiochem Catalog, MerckBiosciences 2006/2007) and (Fmoc Solid Phase Peptide Synthesis, Editedby W. C. Chan and P. D. White, Oxford University Press, 2000, ISBN0-19-963724-5).

One example of an N-terminal extension peptide having the generalformula Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ is;

wherein R1 and R2 are individually chosen protecting groups such as butnot limited to: Carbamates including but not limited to9H-fluoren-9-ylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc) andBenzylcarbarmate (Cbz); Amides including but not limited to benzamide;imides including but not limited to N-Phthalimide; and alkyls includingbut not limited to 1-Adamantyl (1-Adoc) and triphenylmethyl (Trt). In apreferred embodiment R1 is selected from the group consisting of9H-fluoren-9-ylmethoxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc) andbenzylcarbarmate (Cbz), and R2 is selected from the group consisting oftert-butoxycarbonyl (Boc) and triphenylmethyl (Trt). R3 is anindividually chosen protecting group such as but not limited to: Estersincluding but not limited to methyl, ethyl, tert-butyl, benzyl and9H-fluoren-9-ylmethyl (Fm). In a preferred embodiment R3 is tert-butylor benzyl.

An example of an N-terminal extension peptide of the sequenceXaa₇-Xaa₈-Xaa₉-Xaa₁₀ which is activated as the correspondingN-hydroxysuccinimide ester is;

wherein R1, R2, and R3 are individually chosen protecting groups such asdescribed above.

In a further aspect of the invention the N-terminal amino acid extensioncomprising one or more non-proteogenic amino acids has the generalformula

Xaa₇-Xaa₈

whereinXaa₇ is selected from L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine; 1-methylhistidine, 3-methyl histidine,4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine; andXaa₈ is selected from Gly, Aib, (1-aminocyclopropyl) carboxylic acid,(1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylicacid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl) carboxylic acid and alpha-methylproline

The free carboxylic acid of the sequence Xaa₇-Xaa₈ may efficiently beprepared on a resin such as 2-chlorotritylchloride resin e.g. via anFmoc based strategy using adequately protected building blocks usingappropriate reaction conditions as described above or alternatively itmay be prepared in solution. The amino acids in the sequence can beprotected or unprotected.

The N-terminal extension peptide of sequence Xaa₇-Xaa₈ may be activatedto activated acylating agents as described above.

An example of a N-terminal extension peptide with the formula Xaa₇-Xaa₈is:

wherein R1 and R2 are individually chosen protecting groups as describedabove.

An example of an N-terminal extension peptide with the sequenceXaa₇-Xaa₈ which is activated as the corresponding N-hydroxysuccinimideester is;

wherein R1 and R2 are individually chosen protecting groups as describedabove.

In a yet further aspect of the invention, the N-terminal amino acidextension comprising one non-proteogenic amino acid has the generalformula

Xaa₇

whereinXaa₇ is selected from D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine; 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine.

The free carboxylic acid of Xaa₇ may efficiently be prepared on a resinsuch as 2-chlortritylchloride resin using adequately protected buildingblocks using appropriate reaction conditions described as describedabove or alternatively may be prepared in a solution. The amino acid canbe protected or unprotected.

The amino acid in the N-terminal extension Xaa₇ may be activated to anactivated acylating agent as described above.

An example of an amino acid of the formula Xaa₇ is;

wherein R2 is a protecting group as described above or hydrogen.

An example of an amino acid of the formula Xaa₇ which is activated asthe corresponding N-hydroxysuccinimide ester is:

In a further aspect of the invention, the GLP-1 analogue producedaccording to the invention, is a GLP-1 analogue wherein the N-terminalamino acid extension comprising one or more non-proteogenic amino acidshas the general formula Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ as defined above;

and the precursor molecule of said GLP-1 analogue has the generalformulaThr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅as defined above.

In a further aspect of the invention, the GLP-1 analogue producedaccording to the invention, is a GLP-1 analogue wherein the N-terminalamino acid extension comprising one or more non-proteogenic amino acidshas the general formula

Xaa₇-Xaa₈ as defined above;and the precursor molecule of said GLP-1 analogue has the generalformulaXaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅as defined above.

In a yet further aspect of the invention, the GLP-1 analogue producedaccording to the invention, is a GLP-1 analogue wherein the N-terminalamino acid extension comprising one or more non-proteogenic amino acidshas the general formula

Xaa₇ as defined above;and the precursor molecule of said GLP-1 analogue has the generalformulaXaa₈-Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅as defined above.

Coupling of additional amino acid analogues on an unprotectedpolypeptide fragment such as the GLP-1 analogue precursor molecule maynot be a trivial task, due to the large number of possible sidereactions that can take place under the reaction conditions used. Anelectrophilic reagent like for example an active ester such as anN-hydroxysuccinimide ester will in general react with nucleophilespresent in a polypeptide. Such nucleophiles can be thiols, amines,imidazoles, phenolic hydroxy groups, alcohols, and guanidines. The rateof acylation on the functional groups will depend on the inherentreactivity of individual groups but also on the primary, secondary,tertiary and even quaternary structure of the polypeptide. Therefore itwill often be difficult to forecast the relative reactivity of differentamino acid moieties in a polypeptide. In practice reactions on anunprotected polypeptide give rise to mixtures of products. The relativereactivity of the individual groups in a polypeptide can however in somecases partly be controlled by the conditions under which the reaction isperformed. Hence, a change in pH in a reaction mixture can lead toaltered reactivity of some groups. Likewise, a change in the solventfrom a non-protic to a protic one can result in altered reactivity ofsome groups.

In the method according to the invention, the coupling of the N-terminalamino acid extension comprising one or more non-proteogenic amino acidsto the GLP-1 precursor molecule, may in certain useful embodiments takeplace in a solvent, including an organic polar solvent selected from1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide and N-methyl-formamide.

In other useful embodiments, the coupling of the N-terminal amino acidextension may take place in an aqueous solvent mixture, such as anaqueous solvent mixture that may comprise an organic polar solvent, suchas a solvent selected from 1-methyl-pyrrolidin-2-one, acetonitril,tetrahydrofuran, dimethylsulfoxide, N,N-dimethylformamide andN-methyl-formamide.

Furthermore, it has been found that the coupling of the N-terminal aminoacid extension according to the invention, may in useful embodimentstake place in an aqueous solvent mixture, wherein the pH in the reactionmixture is between 6 and 12, such as between 7 and 10. In furtherembodiments the pH in the reaction mixture is in the range of 7-8, 8-9,9-10 or 10-12.

As previously mentioned, the amino acids in the N-terminal amino acidextension comprising one or more non-proteogenic amino acids maycomprise one or more protection groups. Hence, it is contemplated thatthe method according to the invention may further comprise one or moresteps of removing one or more of these protection groups from theN-terminal amino acid extension comprising one or more non-proteogenicamino acids. Such deprotection steps are well described in theliterature.

The method according to the invention may include a further step ofacylating the epsilon-amino-group of at least one lysine residue in theexpressed precursor molecule or the GLP-1 analogue obtained fromcoupling the N-terminal extension molecule to the expressed precursormolecule, with an acylating agent.

In certain embodiments it may be advantageous to conduct this stepbefore the coupling of the N-terminal amino acid extension comprisingone or more non-proteogenic amino acids to the GLP-1 analogue precursormolecule. Hence, the GLP-1 analogue precursor molecule used in themethod according to the invention may in certain embodiments be acylatedwith an acylating agent, optionally activated, on e.g. anepsilon-amino-group of a lysine residue.

Thus, in one aspect the present invention is related to a method formaking a GLP-1 analogue or derivative comprising one or morenon-proteogenic amino acids in the N-terminal part, said methodcomprising the steps of:

(i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue under suitable conditions forexpression of said precursor molecule,(ii) separating the expressed precursor molecule from the culture broth,(iii) optionally acylating the epsilon-amino-group of at least onelysine residue in the expressed precursor molecule with an acylatingagent, which is optionally activated, to obtain a precursor moleculederivative, and optionally isolating said precursor molecule derivative,(iv) coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the expressed precursor molecule orprecursor molecule derivative,(v) isolating the resulting GLP-1 analogue or derivative by suitablemeans.

The method according to the invention may optionally further include astep of selectively protecting the epsilon-amino-group of at least onelysine residue with an amine protecting agent as described above.

In certain embodiments it may be advantageous to conduct this protectionstep prior to the coupling of the N-terminal amino acid extensioncomprising one or more non-proteogenic amino acids to the GLP-1 analogueprecursor molecule. Hence, the resulting lysine-protected GLP-1 analogueprecursor molecule derivative used in the method according to saidembodiment of the invention may be coupled to the N-terminal amino acidextension comprising one or more non-proteogenic amino acids. After saidcoupling of the N-terminal amino acid extension to the lysine-protectedGLP-1 analogue precursor molecule, the protection group(s) on theepsilon amine groups of any lysine(s) may be removed using conventionaltechniques and the resulting epsilon-amino-group of a lysine mayoptionally be acylated with an acylating agent, optionally activated.

Thus, in one aspect the present invention is related to a method formaking a GLP-1 analogue comprising one or more non-proteogenic aminoacids in the N-terminal part, said method comprising the steps of:

(i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said insulinotropic peptide under suitableconditions for expression of said precursor molecule,(ii) separating the expressed precursor molecule from the culture broth,(iii) protecting the epsilon-amino-group of at least one lysine residuein the expressed precursor molecule with a protecting agent to obtain aprotected precursor molecule, and optionally isolating said protectedprecursor molecule,(iv) coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the protected precursor molecule toobtain a protected GLP-1 analogue and optionally isolating saidprotected GLP-1 analogue,(v) deprotection of epsilon amino protection groups, and optionallyisolating the resulting GLP-1 analogue,(vi) optionally acylating the epsilon-amino-group of at least one lysineresidue in the GLP-1 analogue with an acylating agent, which isoptionally activated, to obtain a precursor molecule derivative,(vii) isolating the resulting GLP-1 analogue or derivative by suitablemeans, as known in the art.

In one aspect of the invention, the acylating agent used in theacylation of the epsilon-amino-group of a lysine residue is a carboxylicacid analogue of the general formula:

A-OH, A-C-D-OH, A-B-C—OH or A-B-C-D-OH

wherein one terminal free carboxylic acid, such as the —OH group ofA-OH, A-C-D-OH, A-B-C—OH or A-B-C-D-OH, optionally is activated andwherein any other contained free carboxylic acid(s) optionally areprotected with appropriate protecting group(s) e.g. selected from butnot limited to tert-butyl, benzyl or allyl andwhereinA is selected from the group consisting of;

wherein n is selected from the group consisting of 14, 15, 16 17, 18 and19, p is selected from the group consisting of 10, 11, 12, 13 and 14,and d is selected from the group consisting of 0, 1, 2, 3, 4 and 5, andm is selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, kis selected from the group consisting of 0, 1, 2, 3, 4, 5, 11 and 27,and m is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6,and R1 is selected from the group of 9H-fluoren-9-ylmethoxycarbonyl(Fmoc), tert-butoxycarbonyl (Boc), Benzylcarbamate (Cbz)—B is selected from the group consisting of

wherein x is selected from the group consisting of 0, 1, 2, 3 and 4, andy is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 and 12,C is selected from the group consisting of

wherein b and e are each independently selected from the groupconsisting of 0, 1 and 2, and c and f are each independently selectedfrom the group consisting of 0, 1 and 2 with the proviso that b is 1 or2 when c is 0, or b is 0 when c is 1 or 2, and e is 1 or 2 when f is 0,or e is 0 when f is 1 or 2, and D is selected from the group consistingof

and wherein k is selected from the group consisting of 0, 1, 2, 3, 4, 5,11 and 27, and m is selected from the group consisting of 0, 1, 2, 3, 4,5 and 6.

In one aspect of the invention the fragments A-OH, A-C-D-OH, A-B-C—OH orA-B-C-D-OH used in the acylation of the ε-N of a lysine moiety arecarboxylic acid derivatives that are activated to activated acylatingagents as described above.

In one aspect of the invention a free functional chemical group, such asa contained carboxylic acid in fragment A or C of the fragments A-OH,A-C-D-OH, A-B-C—OH or A-B-C-D-OH is protected with an appropriateprotecting group selected from but not limited to tert-butyl, benzyl orallyl.

When used herein the term “contained” in connection with a peptideand/or an acylating agent about a functional chemical group, such as acarboxylic acid, shall mean a functional chemical group present in thepeptide or acylating agent.

The carboxylic acid derivatives may efficiently be prepared on a resinsuch as 2-chlorotritylchloride resin via an Fmoc based strategy usingadequately protected building blocks using the reaction conditionsdescribed in (Fmoc Solid Phase Peptide Synthesis, Edited by W. C. Chanand P. D. White, Oxford University Press, 2000, ISBN 0-19-963724-5).

An example of a fragment A-B-C-D-OH is:

The corresponding activated analogue could be the N-hydroxysuccinimideester;

Examples of fragments A-B-C-D-:

Examples of fragments A-C-D-:

Examples of fragments A-B-C—:

In all the specific mentioned fragments A-OH, A-C-D-OH, A-B-C—OH orA-B-C-D-OH any contained free carboxylic acid(s), may optionally beprotected with an appropriate protecting group selected from but notlimited to tert-butyl, benzyl or allyl.

In all the specific mentioned fragments A-OH, A-C-D-OH, A-B-C—OH orA-B-C-D-OH the chiral centers whether specified or not may be either theR or S enantiomer independent of other chiral centers in the samefragment.

In one embodiment, when one or more chiral centers are present, thefragments may be in the form of a mixture of enantiomers.

In another embodiment, when one or more chiral centers are present, thefragments may be in the form of a pure enantiomer, wherein each of thechiral centers are either R or S.

In another embodiment, the chirality is as depicted in the specificallymentioned fragments of the present invention.

In one aspect of the invention the epsilon amine protection group of alysine amino acid residue may be selected from, but is not limited to,the following examples:

In the method according to the invention, the acylation step may incertain useful embodiments take place in a solvent, including an organicpolar solvent selected from 1-methyl-pyrrolidin-2-one, acetonitril,tetrahydrofuran, dimethylsulfoxide, N,N-dimethylformamide andN-methyl-formamide.

In a further embodiment, the acylation step may take place in an aqueoussolvent mixture, such as an aqueous solvent mixture comprising anorganic polar solvent. Such organic polar solvent may advantageously beselected from 1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide and N-methyl-formamide.

In a further embodiment, the acylation step may take place in aqueoussolution.

The amount of 1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide and N,N-dimethylformamide or N-methyl-formamide may incertain embodiments be such that the amount of said solvent is less than80% (v/v), such as less than 50% (v/v). In other aspects the amount of1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide or N-methyl-formamide is in therange of about 40-80% (v/v), including the range of 40-45, 45-50, 50-60,60-70 and 70-80% (v/v). It is furthermore contemplated that theacylating agent may be added to the reaction mixture as a solid.

The pH value of the aqueous solvent reaction mixture wherein theacylation step takes place, is in useful embodiments between 9 and 13,such as between 10 and 12, including between 10.5 and 11.5. In furtherembodiments the pH in the reaction mixture is in the range of 9-10,10-11, 11-12 or 12-13.

The acylating agent used in accordance with the invention, may compriseone or more protection groups. Hence, it is contemplated that the methodaccording to the invention may further comprise one or more steps ofremoving one or more of these protection groups from the acylating agentonce it has been coupled to the GLP-1 analogue precursor molecule. Suchdeprotection steps are well described in the literature.

Efficient heterologous protein expression in host cells, often requirethat the protein is expressed in the form of a pro-peptide, i.e. apolypeptide having a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence). However, this leadersequence is normally not wanted in the desired polypeptide, andtherefore the method according to the invention may in certain aspectsinclude a further step of removing N-terminal and/or C-terminalpro-peptide extensions from the GLP-1 analogue precursor molecule.

The method according to the invention comprises a step of isolating theresulting GLP-1 analogue or derivative by suitable means, e.g. standardpurification methods known in the art.

It is also within the scope of the invention that the GLP-1 analogues orderivatives resulting from the method according to the invention incertain embodiments may be derivatized with an albumin binding moiety orbe pegylated.

The leader sequences of the present invention may be in the optimizedform as listed in FIG. 4.

The term “albumin binding moiety” as used herein means a residue whichbinds non-covalently to human serum albumin. The albumin binding residueattached to the therapeutic polypeptide typically has an affinity below10 μM to human serum albumin and preferably below 1 μM. A range ofalbumin binding residues are known among linear and branchedlipohophillic moieties containing 4-40 carbon atoms having a distalacidic group.

EMBODIMENTS ACCORDING TO THE INVENTION

1. A method for making a GLP-1 analogue or derivative comprising one ormore non-proteogenic amino acids in the N-terminal part, said methodcomprising the steps of

(i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue under suitable conditions forexpression of said precursor molecule,(ii) separating the expressed precursor molecule from the culture broth,(iii) coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the expressed precursor molecule,(iv) isolating the resulting GLP-1 analogue or derivative by suitablemeans.

2. A method for making a GLP-1 analogue or derivative comprising one ormore non-proteogenic amino acids in the N-terminal part, said methodcomprising the steps of:

i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue under suitable conditions forexpression of said precursor molecule,ii) separating the expressed precursor molecule from the culture broth,iii) protecting the lysine group(s) of the precursor molecule,iv) coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the protected precursor molecule, toobtain a protected GLP-1 analogue,v) deprotecting the lysine group(s) of the said protected GLP-1analogue,vi) isolating the resulting GLP-1 analogue or derivative by suitablemeans.

3. A method according to embodiments 1 or 2, wherein the N-terminalamino acid extension is protected before its use in the coupling step,where an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids is coupled to the protected precursormolecule, and wherein the protected GLP-1 analogue is deprotected againafter coupling of the N-terminal extension.

4. A method for making a GLP-1 analogue or derivative comprising one ormore non-proteogenic amino acids in the N-terminal part, said methodcomprising the steps of:

i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue under suitable conditions forexpression of said precursor molecule,ii) separating the expressed precursor molecule from the culture broth,iii) coupling a protected N-terminal amino acid extension comprising oneor more non-proteogenic amino acids to the expressed precursor molecule,iv) deprotecting any protecting group of the protected GLP-1 analogue orderivative,v) isolating the resulting GLP-1 analogue or derivative by suitablemeans.

5. A method for making a GLP-1 analogue or derivative comprising one ormore non-proteogenic amino acids in the N-terminal part, said methodcomprising the steps of:

i) culturing a host cell comprising a nucleotide sequence encoding aprecursor molecule of said GLP-1 analogue under suitable conditions forexpression of said precursor molecule,ii) separating the expressed precursor molecule from the culture broth,iii) protecting the lysine group(s) of the precursor molecule,iv) coupling a protected N-terminal amino acid extension comprising oneor more non-proteogenic amino acids to the protected precursor molecule,to obtain a protected GLP-1 analogue or derivative,v) deprotecting any protecting group of the protected GLP-1 analogue orderivative,vi) isolating the resulting GLP-1 analogue or derivative by suitablemeans.

6. A method according to any of the previous embodiments, wherein theprecursor molecule of said GLP-1 analogue is selected from the list ofprecursor molecules comprising the amino acid sequence of the generalformula:

Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅wherein

Xaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr or Gln;Xaa₂₀ is Leu or Met; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys or Arg;Xaa₂₅ is Ala or Val; Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu;Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu,Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆ is Arg, Gly or Lys;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or is absent;Xaa₃₈ is Lys, Ser, or is absent.Xaa₃₉ is Ser, Lys, or is absent;Xaa₄₀ is Gly, or is absent;Xaa₄₁ is Ala, or is absent;Xaa₄₂ is Pro, or is absent;Xaa₄₃ is Pro, or is absent;Xaa₄₄ is Pro, or is absent;Xaa₄₅ is Ser, or is absent;provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ orXaa₄₅ is absent then each amino acid residue downstream is also absent.

7. A method according to any of embodiments 1-5, wherein the precursormolecule of said GLP-1 analogue is selected from the list of precursormolecules comprising the amino acid sequence of the general formula:

Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃- Xaa₄₄-Xaa₄₅wherein

Xaa₉ is Glu or Asp Xaa₁₀ is Gly or Ala Xaa₁₆ is Val or Leu; Xaa₁₈ isSer, Lys or Arg; Xaa₁₉ is Tyr or Gln; Xaa₂₀ is Leu or Met; Xaa₂₂ is Glyor Glu; Xaa₂₃ is Gln, Glu, Lys or Arg; Xaa₂₅ is Ala or Val; Xaa₂₆ isLys, Glu or Arg; Xaa₂₇ is Glu or Leu; Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ isVal, Lys or Arg; Xaa₃₄ is Lys, Glu, Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆is Arg, Gly or Lys;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or is absent;Xaa₃₈ is Lys, Ser, or is absent.Xaa₃₉ is Ser, Lys, or is absent;Xaa₄₀ is Gly, or is absent;Xaa₄₁ is Ala, or is absent;Xaa₄₂ is Pro, or is absent;Xaa₄₃ is Pro, or is absent;Xaa₄₄ is Pro, or is absent;Xaa₄₅ is Ser, or is absent;provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ orXaa₄₅ is absent then each amino acid residue downstream is also absent.

8. A method according to any of embodiments 1-5, wherein the precursormolecule of said GLP-1 analogue is selected from the list of precursormolecules comprising the amino acid sequence of the general formula:

Xaa₈-Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃- Xaa₄₄-Xaa₄₅wherein

Xaa₈ is Ala, Gly, Val, Leu, Ile, Lys Xaa₉ is Glu, Asp Xaa₁₀ is Gly, AlaXaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr or Gln;Xaa₂₀ is Leu or Met; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys or Arg;Xaa₂₅ is Ala or Val; Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu;Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu,Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆ is Arg, Gly or Lys;

Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or is absent;Xaa₃₈ is Lys, Ser, or is absent.Xaa₃₉ is Ser, Lys, or is absent;Xaa₄₀ is Gly, or is absent;Xaa₄₁ is Ala, or is absent;Xaa₄₂ is Pro, or is absent;Xaa₄₃ is Pro, or is absent;Xaa₄₄ is Pro, or is absent;Xaa₄₅ is Ser, or is absent;provided that if Xaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ orXaa₄₅ is absent then each amino acid residue downstream is also absent.

9. A method according to any of the previous embodiments, wherein thenon-proteogenic amino acids in the N-terminal amino acid extensioncomprising one or more non-proteogenic amino acids, are selected fromthe group consisting of γ-carboxyglutamate, ornithine, phosphoserine,D-amino acids such as D-alanine and D-glutamine, D-alanine andD-leucine, Aib (α-aminoisobutyric acid), Abu α-aminobutyric acid), Tle(tert-butylglycine), 3-aminomethyl benzoic acid, anthranilic acid,des-amino-Histidine, β-alanine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,Nα-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine, 4-pyridylalanine,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid,(1-aminocyclooctyl) carboxylic acid, α-methyl prolin, 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine.

10. A method according to any of the previous embodiments, wherein thelength of the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids is 1 amino acid.

11. A method according to any of embodiments 1-9, wherein the length ofthe N-terminal amino acid extension comprising one or morenon-proteogenic amino acids is 2 amino acids.

12. A method according to any of embodiments 1-9, wherein the length ofthe N-terminal amino acid extension comprising one or morenon-proteogenic amino acids is 3 amino acids.

13. A method according to any of embodiments 1-9, wherein the length ofthe N-terminal amino acid extension comprising one or morenon-proteogenic amino acids is 4 amino acids.

14. A method according to any of the previous embodiments, wherein theN-terminal amino acid extension comprises two non-proteogenic aminoacids.

15. A method according to any of embodiments 1-13, wherein theN-terminal amino acid extension comprises three non-proteogenic aminoacids.

16. A method according to any of embodiments 1-13, wherein theN-terminal amino acid extension comprises four non-proteogenic aminoacids.

17. A method according to any of the previous embodiments, wherein theN-terminal amino acid extension comprising one or more non-proteogenicamino acids has the general formula

Xaa₇-Xaa₈-Xaa₉-Xaa₁₀

whereinXaa₇ is selected from L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine, 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acid8-(1,2,4-triazol-1-yl)-alanineXaa₈ is selected from Ala, Gly, Val, Leu, Ile, Lys, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid and alpha-methyl prolineXaa₉ is selected from Glu, Asp, γ,γ-dimethyl Glu, β,β-dimethyl Glu andβ,β-dimethyl Asp, andXaa₁₀ is selected from Gly, Aib, (1-aminocyclopropyl) carboxylic acid,(1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylicacid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)carboxylic acid, and (1-aminocyclooctyl) carboxylic acid.

18. A method according to any of embodiments 1-16, wherein theN-terminal amino acid extension comprising one or more non-proteogenicamino acids has the general formula

Xaa₇-Xaa₈whereinXaa₇ is selected from L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine; 1-methylhistidine, 3-methyl histidine,4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine; andXaa₈ is selected from Gly, Aib, (1-aminocyclopropyl) carboxylic acid,(1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylicacid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)carboxylic acid, or (1-aminocyclooctyl) carboxylic acid and alpha-methylproline

19. A method according to any of embodiments 1-16, wherein theN-terminal amino acid extension comprising one non-proteogenic aminoacid having the general formula

Xaa₇whereinXaa₇ is selected from D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine; 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine.

20. A method according to embodiment 19, wherein the N-terminal aminoacid extension is desamino-histidine and the coupling reaction iscarried out by a4-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)-ethyl]-1H-imidazol-1-iumsalt, such as trifluoroacetate, with the expressed precursor molecule.

21. A method according to any of embodiments 1-5, wherein the N-terminalamino acid extension comprising one or more non-proteogenic amino acidshas the general formula

Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ as defined in embodiment 17;and the GLP-1 analogue has the general formulaThr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅as defined in embodiment 3.

22. A method according to any of embodiments 1-5, wherein the N-terminalamino acid extension comprising one or more non-proteogenic amino acidshas the general formula

Xaa₇-Xaa₈ as defined in embodiment 18;and the GLP-1 analogue has the general formulaXaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅as defined in embodiment 4.

23. A method according to any of embodiments 1-5, wherein the N-terminalamino acid extension comprising one or more non-proteogenic amino acidshas the general formula

Xaa₇ as defined in embodiment 19;and the GLP-1 analogue has the general formulaXaa₈-Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅as defined in embodiment 4.

24. A method according to any of embodiments 17-23, wherein theC-terminal non-proteogenic or proteogenic amino acid in the N-terminalextensions Xaa₇-Xaa₈-Xaa₉-Xaa₁₀ or Xaa₇-Xaa₈, or the non-proteogenicamino acid Xaa₇ is activated.

25. A method according to embodiment 24, wherein the activated aminoacid(s) are selected from the group consisting of: acid chloride, acidbromide, acid fluoride, symmetrical anhydride, mixed anhydride,carboxylic acids activated using common carbodiimides, carboxylic acidsusing carbodiimides and an additive and carboxylic acids activated withan uronium salt or a phosphonium salt.

26. A method according to embodiment 24, wherein the activated aminoacid(s) are selected from the group consisting of: carboxylic acidsactivated using common carbodiimides, diisopropylcarbodiimide (DIPCDI),N,N′-dicyclohexylcarbodiimide (DCC),1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).

27. A method according to embodiment 24, wherein the activated aminoacid(s) are selected from the group consisting of: carboxylic acidsusing carbodiimides and an additive selected from the group consistingof N-hydroxybenzotriazol (HOBt), 1-Hydroxy-7-azabenzotriazole,6-chloro-N-hydroxybenzotriazol (HOAt) and3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (DhbtOH),

28. A method according to embodiment 24, wherein the activated aminoacid(s) are selected from the group consisting of: carboxylic acidsactivated with an uronium salt or a phosphonium salt,O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU), O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TBTU),2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate (HATU),1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate(BOP) benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate(PYBOP), esters of N-hydroxysuccinimid (NHS ester), pentafluorophenolester (PfP-ester), 2,4-dinitrophenyl ester, 4-nitrophenyl ester,3-Hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt)Carbonyldiimidazole (CDI), N-ethyl-5-phenylisoxazolium-3′-sulfonate(NEPIS).

29. A method according to any of the previous embodiments, wherein thecoupling of the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids takes place in an organic polar solventselected from the group consisting of 1-methyl-pyrrolidin-2-one,acetonitril, tetrahydrofuran, dimethylsulfoxide N,N-dimethylformamideand N-methyl-formamide.

30. A method according to any of the previous embodiments, wherein thecoupling of the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids takes place in an aqueous solvent mixture.

31. A method according to embodiment 30, wherein the aqueous solventmixture comprises an organic polar solvent selected from the groupconsisting of 1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide N,N-dimethylformamide and N-methyl-formamide.

32. A method according to embodiments 30 or 31, wherein the coupling ofthe N-terminal amino acid extension comprising one or morenon-proteogenic amino acids takes place in an aqueous solvent mixture,wherein the pH in the reaction mixture is between 4 and 12, preferablybetween 6 and 10.

33. A method according to any of the previous embodiments, wherein theamino acids in the N-terminal amino acid extension comprising one ormore non-proteogenic amino acids comprises one or more protectiongroups.

34. A method according to embodiment 33, further comprising the step ofremoving one or more protection groups from said N-terminal amino acidextension comprising one or more non-proteogenic amino acids.

35. A method according to embodiment 1, further comprising the step ofacylating the epsilon-amino-group of at least one lysine residue in theexpressed precursor molecule with an acylating agent.

36. A method according to embodiment 35, wherein the acylating agent isa carboxylic acid analogue of the general formula;

A-OH, A-C-D-OH, A-B-C—OH or A-B-C-D-OH

which is optionally activated and/or protected with one or moreprotection group(s),whereinA is selected from the group consisting of;

wherein n is selected from the group consisting of 14, 15, 16 17, 18 and19, p is selected from the group consisting of 10, 11, 12, 13 and 14,and d is selected from the group consisting of 0, 1, 2, 3, 4 and 5, andm is selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, kis selected from the group consisting of 0, 1, 2, 3, 4, 5, 11 and 27,and m is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6,and R1 is selected from the group of 9H-fluoren-9-ylmethoxycarbonyl(Fmoc), tert-butoxycarbonyl (Boc), Benzylcarbarmate (Cbz).B is selected from the group consisting of

wherein x is selected from the group consisting of 0, 1, 2, 3 and 4, andy is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 and 12,C is selected from the group consisting of

wherein b and e are each independently selected from the groupconsisting of 0, 1 and 2, and c and f are each independently selectedfrom the group consisting of 0, 1 and 2 with the proviso that b is 1 or2 when c is 0, or b is 0 when c is 1 or 2, and e is 1 or 2 when f is 0,or e is 0 when f is 1 or 2, andD is selected from the group consisting of

and wherein k is selected from the group consisting of 0, 1, 2, 3, 4, 5,11 and 27, and m is selected from the group consisting of 0, 1, 2, 3, 4,5 and 6.

37. A method according to embodiment 36, wherein the carboxylic acidA-OH, A-C-D-OH, A-B-C—OH or A-B-C-D-OH is activated to activatedacylating agent.

38. A method according to embodiment 35, wherein said acylation steptakes place in an aqueous solvent mixture.

39. A method according to embodiment 38, wherein said aqueous solventmixture comprises an organic polar solvent.

40. A method according to embodiment 39, wherein said organic polarsolvent is selected from the group consisting of1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide and N-methyl-formamide.

41. A method according to embodiment 40, wherein the amount of1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide or N-methyl-formamide is lessthan 80% (v/v).

42. A method according to embodiment 40, wherein the amount of1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide or N-methyl-formamide is lessthan 50% (v/v).

43. A method according to embodiment 40, wherein the amount of1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide, N,N-dimethylformamide or N-methyl-formamide is in therange of about 40-80% (v/v).

44. A method according to embodiment 35, wherein the acylating agent isadded to the reaction mixture as a solid.

45. A method according to embodiment 35, wherein said acylation steptakes place in an aqueous solvent mixture, wherein the pH in thereaction mixture is between 9 and 13, preferably between 10 and 12 oreven more preferably between 10.5 and 11.5.

46. A method according to embodiment 35, wherein the acylating agent,which is optionally activated, comprises one or more protection groups.

47. A method according to embodiment 46, further comprising the step ofremoving one or more protection groups from said activated acylatingagent.

48. A method according to any of the previous embodiments, furthercomprising the step of removing an N-terminal pro-peptide extension fromthe resulting precursor molecule.

49. A method according to any of embodiments 1-47, further comprisingthe step of removing a C-terminal pro-peptide extension from theresulting precursor molecule.

50. A method according to any of the previous embodiments, wherein thehost cell is a mammalian host cell.

51. A method according to any of embodiments 1-49, wherein the host cellis a bacterial host cell.

52. A method according to any of embodiments 1-49, wherein the host cellis a yeast host cell.

53. A method according to embodiment 52, wherein the yeast host cell isSaccharomyces cerivisiae.

54. A method according to any of the previous embodiments, wherein theGLP-1 analogue is an analogue or derivative of human GLP-1(7-37), humanGLP-2, exendin-3 or exendin-4.

55. A method according to any of the previous embodiments, wherein theGLP-1 analogue is an analogue or derivative of human GLP-1(7-37).

56. A method according to any of the previous embodiments, wherein saidGLP-1 analogue is selected from the group consisting ofAib⁸-GLP-1(7-36)-amide, Aib⁸-GLP-1(7-37), Aib^(8,35) GLP-1(7-37),[Aib⁸,Arg³⁴]GLP-1-(7-37),

-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [DesaminoHis⁷,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [DesaminoHis⁷,Glu²²,Arg²⁶,Glu³⁰,Arg³⁴,Lys³⁷]GLP-1-(7-37),-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1(7-37)amide,-   [DesaminoHis⁷,Arg³⁴]GLP-1-(7-37),    [Aib⁸,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,-   [DesaminoHis⁷, Glu²² Arg²⁶, Arg³⁴, Phe(m-CF3)²⁸]GLP-1-(7-37)amide,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴]GLP-1-(7-37)-Lys,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴]GLP-1-(7-37)-Lys,-   [dDesaminoHis⁷,Arg²⁶,Arg³⁴,]GLP-1-(7-37)-Lys,-   [DesaminoHis⁷,Glu²²,Arg²⁶,Arg³⁴,Lys³⁷]GLP-1-(7-37)amide,    and analogues thereof.

57. A method according to any of the previous embodiments, wherein theGLP-1 analogue is the only biologically active substance in thepharmaceutical composition.

58. A method according to any of the previous embodiments, wherein theGLP-1 analogue is an exendin-4 analogue.

59. The method according to any of the previous embodiments comprising astep of selectively protecting the epsilon-amino-group of at least onelysine residue.

60. The method according to embodiment 59 wherein the step is addedafter step (ii) separating the expressed precursor molecule from theculture broth.

61. The method according to embodiment 59 wherein the step is addedafter step (iv) as defined in embodiment 1 isolating the resulting GLP-1analogue or derivative by suitable means.

62. The method according to embodiment 19, wherein the N-terminal aminoacid is desamino-histidine introduced by the activated acylating agent4-{3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl}-1Himidazol-1-ium

63. The method according to embodiment 19, wherein the N-terminal aminoacid is desamino-histidine introduced by the use of the activatedacylated agent4-{3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl}-1Himidazol-1-iumtrifluoroacetate

64. The method according to any of the previous embodiments, wherein thecoupling of the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids takes place in an organic polar solventselected from the group consisting of 1-methyl-pyrrolidin-2-one,acetonitril, tetrahydrofuran, dimethylsulfoxide N,N-dimethylformamideand N-methyl-formamide.

65. The method according to any of embodiments 1-63, wherein thecoupling of the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids takes place in an aqueous solvent mixture.

66. The method according to embodiment 65, wherein the aqueous solventmixture comprises an organic polar solvent selected from the groupconsisting of 1-methyl-pyrrolidin-2-one, acetonitril, tetrahydrofuran,dimethylsulfoxide N,N-dimethylformamide and N-methyl-formamide.

67. The method according to any of embodiments 1-63, wherein thecoupling of the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids takes place in an aqueous solvent mixture,wherein the pH in the reaction mixture is between 7 and 11, preferablybetween 8 and 10.

68. A method of introducing des-amino histidine in a insulinotropicagent with the use of the activated acylating agent4-{3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl}-1Himidazol-1-ium:

69. A method of introducing des-amino histidine in a insulinotropicagent with the use of the activated acylated agent4-{3-[(2,5-dioxo-1-pyrrolidinyl)oxy]-3-oxopropyl}-1Himidazol-1-iumtrifluoroacetate

The present invention will now be described in more details in thefollowing non-limiting Examples and Drawings.

EXAMPLES Abbreviations Used

-   Adoc: adamantyloxycarbonyl-   Aib: α-aminoisobutyric acid-   Boc: tert butyloxycarbonyl-   CH₃CN: acetonitrile-   DCM: dichloromethane-   DIC: Diisopropylcarbodiimide-   DIPEA: diisopropylethylamine-   DMF: N,N dimethylformamide-   EtOAc: Ethylacetate-   Et₂O: diethylether-   Fmoc: 9H-fluoren-9-ylmethoxycarbonyl-   HATU: 2-(7-azabenzotriazol-1-yl-)-1,1,3,3 tetramethyluronium    hexafluorophosphate-   HBTU: 2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluronium    hexafluorophosphate-   H₂O: water-   HOAt: 1-Hydroxy-7-azabenzotriazole-   HOBt: 1-Hydroxybenzotriazole-   MeCN: acetonitrile-   Mtt: 4-Methyltrityl-   MW: Molecular weight-   NaOH: Sodium hydroxide-   NHS: N-Hydroxysuccinimide-   NMP: 1-Methyl-pyrrolidin-2-one-   OtBu: tert butyl ester-   PyBoP (benzotriazol-1-yloxy)tripyrrolidinophosphonium    hexafluorophosphate-   PyBroP bromo-tris-pyrrolidinophosphonium hexafluorophosphate-   r.t: room temperature-   OSu: N-Hydroxysuccinimide ester-   tBu: tert-butyl-   TBME: tert-butylmethylether-   TEA: triethylamine-   TFA: trifluoroacetic acid-   TFFH Tetramethylfluoroformamidinium hexafluorophosphate-   TIPS: triisopropylsilane-   Trt: Trityl, triphenylmethyl-   TSTU: O—(N-succinimidyl)-1,1,3,3-tetramethyluronium    tetrafluoroborate

General Methods General Method for the Preparation of Peptides (MethodA)

The peptide can be synthesized according to the Fmoc strategy on anApplied Biosystems 433 peptide synthesizer in 0.25 mmol or 1.0 mmolscale using the manufacturer supplied FastMoc UV protocols which employHBTU (2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate) or HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) mediated couplings in NMP (N-methylpyrrolidone),and UV monitoring of the deprotection of the Fmoc protection group. Wangor trityl based resins can be used as solid support and the protectedamino acid derivatives used is standard Fmoc-amino acids (supplied frome.g. Anaspec, or Novabiochem) supplied in preweighed cartridges suitablefor the ABI433A synthesizer. The N terminal amino acid is Boc protectedat the alpha amino group (e.g. Boc-His(Boc)OH is used for peptides withHis at the N-terminal). The synthesis of the peptides may in some casesbe improved by the use of dipeptides protected on the dipeptide amidebond with a group that can be cleaved under acidic conditions such butnot limited to 2-Fmoc-oxy-4-methoxybenzyl or 2,4,6-trimethoxybenzyl. Incases where a serine or a threonine is present in the peptide, the useof pseudoproline dipeptides may be used (see e.g. catalogue fromNovobiochem 2002/2003 or newer version, or W.R. Sampson (1999), J. Pep.Sci. 5, 403.

General Method for the Preparation of Peptides (Method B)

One alternative method of peptide synthesis is by Fmoc chemistry on amicrowave-based Liberty peptide synthesizer (CEM Corp., North Carolina).Wang or trityl based resins can be used as solid support The couplingchemistry is performed with DIC/HOAt in NMP using amino acid solutionsof 0.4 M in NMP and a molar excess of 8-10 fold. Coupling conditions is5 minutes at up to 70° C. Deprotection is achieved with 5% piperidine inNMP at up to 70° C.

The GLP-1 precursor molecules can e.g. be purified by a variety ofchromatographic procedures, e.g. ion exchange chromatography,hydrophobic interaction chromatography, gel filtration chromatography,affinity chromatography, reverse phase HPLC or the like, dependent onthe type of polypeptide in question.

General Method for the Preparation of Acylating Agents A-OH A-B-C-D-OH,A-C-D-OH, A-B-C—OH and N-Terminal Amino Acid ExtensionsXaa₇-Xaa₈-Xaa₉-Xaa₁₀, Xaa₇-Xaa₈ and Xaa₇

The necessary carboxylic acids can be prepared on a2-chlorotritylcloride resin using standard Fmoc chemistry. First Fmocprotected amino carboxylic acid can be attached to the2-chlorotritylchloride resin by swelling the resin in a suitable solventlike NMP, DCM, DMF or the like, preferably DCM. The adequately protectedFmoc amino carboxylic acid is added together with a suitable base suchas DIPEA or TEA, and the resin is agitated for a suitable period of timesuch as 30 min at r.t. The resin is washed with a suitable solvent suchas NMP, DMF or DCM.

Fmoc deprotection is achieved using piperidine in NMP, preferably 20%piperidine in NMP at r.t. for 1 to 30 min, typically 10 min, before theresin is washed thoroughly with NMP, DMF or DCM. The step is repeateduntil complete deprotection is obtained, typically 3 times or more.

Coupling of the next Fmoc protected amino carboxylic acid is achievedusing standard coupling conditions; the resin is swelled in a solventlike NMP, DMF, DCM or a mixture of these. Another solution of Fmocprotected amino carboxylic acid in a solvent like NMP, DMF, DCM or amixture of these and HOBt or HOAt is added DIC or an equivalent of such.This mixture is added to the resin and the mixture is added DIPEA or TEAand the mixture is agitated at r.t. for 1 to 16 hours typically 3 hours.Alternatively, the mixture is agitated for 1 to 60 min before DIPEA orTEA is added to the mixture. If the coupling is not completed judged byTNBS test, ninhydrine test or choranil test the step is repeated untilnegative test.

Alternatively activation of the Fmoc protected amino carboxylic acid canbe achieved using the following coupling reagents; PyBOP, PyBrOP, HBTU,HATU, or TFFH.

Further incorporation of adequately protected Fmoc-amino carboxylicacids can be introduced using the procedure given above. The lastcoupling step comprising incorporation of a carboxylic acid derivativeis introduced using the coupling conditions described above.

Cleavage from the resin can be achieved by treatment of the resin with20% trifluoroethanol in DCM, DCM-TIPS-TFA (95.5:2.5:2) orhexafluoroisopropanol from 5 min to 3 hours to give the fully protectedderivative.

Cleavage from the resin can also be achieved using TFA-TIPS-Water(95:2.5:2.5) from 5 min to 3 hours to give the fully deprotectedderivatives.

Alternatively the derivatives can be prepared in solution using standardprocedures described in the literature.

Appropriately protected building blocks are commercially available orcan be prepared using procedures described in the literature.

Xaa₇ is commercially available or can be prepared via proceduresdescribed in the literature.

General Method for Activation of Acylating Agents A-OH A-B-C-D-OH,A-C-D-OH, A-B-C—OH and N-Terminal Amino Acid ExtensionsXaa₇-Xaa₈-Xaa₉-Xaa₁₀ Xaa₇-Xaa₈ and Xaa₇

Activation of the required amino acids can be achieved using standardprocedures described in the literature. If an active ester such as anN-hydroxy succinimid ester is required the carboxylic acid is dissolvedin an appropriate solvent such as THF, ethyl acetate, NMP or DMF. Areagent such as O—(N-succinimidyl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate is added to the solution together with a base suchas DIPEA or TEA. The mixture is stirred at r.t. until the reaction iscomplete typically from 3 to 16 hours. Alternatively the carboxylic acidcan be dissolved together with N-hydroxy succinimide and treated withDIC. The product mixture can be used directly without furtherpurification or it can be subjected an aqueous work up.

Purification

The crude peptide was purified by preparative HPLC on a column packedwith C18 silica. After drying the crude peptide was dissolved in 50%acetic acid in H₂O, diluted with H₂O and injected on the column whichthen was eluted with a gradient of CH₃CN in water and 0.1% TFA 10 ml/minduring 50 min at 40° C. The fractions containing the peptide werecollected and lyophilized after dilution with water.

Analysis LCMS

LCMS was performed on a setup consisting of Sciex API 100 Singlequadropole mass spectrometer. The instrument control and dataacquisition were done by the Sciex Sample control software running on aWindows 2000 computer. The HPLC pump is connected to two eluentreservoirs containing:

A: 0.05% Trifluoro acetic acid in waterB: 0.05% Trifluoro acetic acid in acetonitrile

The analysis was performed at room temperature by injecting anappropriate volume of the sample (preferably 20 μl) onto the columnwhich was eluted with a gradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following table:

Column: Waters Xterra MS C-18×3 mm id 5 μm

Gradient: 5%-90% acetonitrile linear during 7.5 min at 1.5 ml/minDetection: 210 nm (analogue output from DAD)ELS (analogue output from ELS): 40° C.MS ionisation mode: API-ES

HPLC

Method 01_B4_(—)2: RP-analysis was performed using a Waters 600S systemfitted with a Waters 996 diode array detector. UV detections at 214 nmand 254 nm were collected using a Symmetry 300 C18, 5 μm, 3.9 mm×150 mmcolumn, 42° C. Eluted with a linear gradient of 5-95% acetonitrile,90-0% water, and 5% trifluoroacetic acid (1.0%) in water over 15 minutesat a flow-rate of 1.0 min/min.

Method 02_B4_(—)4: The RP-analyses was performed using a Alliance Waters2695 system fitted with a Waters 2487 dualband detector. UV detectionsat 214 nm and 254 nm were collected using a Symmetry300 C18, 5 um, 3.9mm×150 mm column, 42° C. Eluted with a linear gradient of 5-95%acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in waterover 15 minutes at a flow-rate of 1.0 min/min.

Example 1 Construction of Yeast Expression Systems and Production ofGLP-1 Analogue Precursor Molecules (R34)GLP-1(8-37)

Expressions plasmids are of the C—POT type, similar to those describedin EP 171,142. These are 2μ-based expression vectors characterized bycontaining the Schizosaccharomyces pombe triose phosphate isomerase gene(POT) for the purpose of plasmid selection and stabilization in S.cerevisiae. The plasmids also contain the S. cerevisiae triose phosphateisomerase promoter and terminator (FIG. 1). These sequences are similarto the corresponding sequences in plasmid pKFN1003 (described in WO90100075) as are all other sequences except the following: 1) Thesequence of the EcoRI-XbaI fragment encoding the fusion protein of theleader and the insulin product. 2) A silent mutation has been introducedresulting in removal of a NcoI-site in the 2μ-region in the expressionvector. In order to facilitate cloning of different fusion proteins athe DNA sequence encoding the MFα1 pre-pro leader has been changed toincorporate a NcoI site (see FIG. 2) and is called the MFα1* pre-proleader. Thus the NcoI-XbaI fragment is simply replaced by an NcoI-XbaIfragment encoding the GLP-1 precursor molecule of interest. SuchNcoI-XbaI fragments may be synthesized using synthetic oligonucleotidesand PCR according to standard techniques. In addition to thealpha-leader other leaders can be used.

Synthetic DNA fragments containing sequences encoding (R34)GLP-1(11-37),(E22,R26,R34)GLP-1(8-37)K38 and (E22,R26,R34,K37)GLP-1(8-37) wereobtained form Geneart AG, BioPark, Josef-Engert-Str. 11, D-93053Regensburg, Germany. The synthetic DNA encoding(E22,R26,R34)GLP-1(8-37)K38 and (E22,R26,R34,K37)GLP-1(8-37) wasfurnished with 3′ and 5′ DNA sequences encoding N- and C-terminalextension to facilitate expression in yeast (FIG. 2) and termedExt1-(E22,R26,R34)GLP-1(8-37)K38-Ext2 andExt1-(E22,R26,R34,K37)GLP-1(8-37)-Ext2. The synthetic DNA was digestedwith NcoI and XbaI and ligated to the NcoI-XbaI vector fragment of themodified cPOT type expression vector (FIG. 1). This resulted in threeexpression plasmids pSA273, pSA277 and pSA278 encoding (R34)GLP-1(11-37)(SEQ ID NO:6), Ext1-(E22,R26,R34)GLP-1(8-37)K38-Ext2 (SEQ ID NO:7) andExt1-(E22,R26,R34,K37)GLP-1(8-37)-Ext2 (SEQ ID NO:8), respectively.

The expression plasmids were propagated in E. coli, grown in thepresence of ampicillin and isolated using standard techniques (Sambrooket al., 1989). The plasmid DNA was checked for insert by appropriaterestriction nucleases (e.g. EcoRI, NcoI, XbaI) and was shown by sequenceanalysis to contain the proper sequence of the GLP-1 analogue precursorsmolecules (R34)GLP-1(11-37), Ext1-(E22,R26,R34)GLP-1(8-37)K38-Ext2 andExt1-(E22,R26,R34,K37)GLP-1(8-37)-Ext2. (FIG. 2).

The plasmids were transformed into S. cerevisiae strain ME1719 (MATa/αleu2/leu2 pep-4-3/pep-4-3 Δtpi::LEU2/Δtpi::LEU2 Δura3/Δura3Δyps1::URA3/Δyps1::ura3 Cir+). This strain is described in WO 98/01535.Yeast transformants harbouring the plasmid were selected by glucoseutilization as carbon source on YPD (1% yeast extract, 2% peptone, 2%glucose) agar (2%) plates. This resulted in three yeast strains: γSA251,γSA259 and γSA260 expressing (R34)GLP-1(11-37) (SEQ ID NO:9),Ext1-(E22,R26,R34)GLP-1(8-37)K38-Ext2 (SEQ ID NO:10) andExt1-(E22,R26,R34,K37)GLP-1(8-37)-Ext2 (SEQ ID NO:11), respectively.

The ME1719 yeast strains γSA251, γSA259 and γSA260 were inoculated into5 ml of growth media, for example a medium consisting of 5 g/L(NH₄)₂SO₄, 184 mg/L (NH₄)₂HPO₄, 2.88 g/L KH₂PO₄, 1.42 g/L MgSO₄.₇H₂O,1.28 g/L, K₂SO₄, 10.00 g/L succinic acid, 10.00 g/L casamino acids,0.0112 g/L FeSO₄. 7H₂O, 0.0086 g/L MnSO₄.H₂O, 0.0014 g/L CuSO₄.5H₂O,0.00185 g/L ZnSO₄.7H₂O, 0.0129 g/L CaCl2.2H₂O, 0.071 g/L citric acid,28.0 mg/L m-inositol, 14.0 mg/L choline chloride, 2.8 mg/L thiamine, 2.8mg/L niacinamide, 2.1 mg/L Ca-pantothenic acid, 0.14 mg/L biotin, 0.14mg/L folic acid, 40 g/L glucose. The cultivation was carried out at 30°C. for 3 days. After centrifugation the supernatant was removed forquantitative HPLC analysis by which method the concentration of secretedGLP-1 analogue was measured. The identity of the GLP-1 precursormolecules was confirmed by LC/MS analysis. The N- and C-terminalextensions employed to facilitate expression of(E22,R26,R34)GLP-1(8-37)K38 and (E22,R26,R34,K37)GLP-1(8-37) wereremoved by the lysine specific Achromobactor Lyticus Protease 1according to well-established procedures.

Example 2 Construction of Yeast Expression System and Production ofGLP-1 Analogue Precursor Molecule (R34)GLP-1(9-37)

A synthetic DNA fragment encoding (R34)GLP-1(9-37) was constructed bystandard PCR using the primers5′-AGGGGTATCCATGGCTAAGAGAGAAGGTACCTTCACCTCTGAC-3′ and5′-AATCTTAGTTTCTAGAGCCTGCG-3′ and a plasmid containing DNA sequenceencoding (R34)GLP-1(7-37). The primers were designed an 5′ NcoI site anda 3′ XbaI site. Thus the PCR fragment could be digested by NcoI and XbaIafter purification and ligated to the NcoI-XbaI vector fragment of themodified cPOT type expression vector described in example 1. Theresulting plasmid encoding (R34)GLP-1(9-37) was named pSA82.

The expression plasmid was propagated in E. coli, grown in the presenceof ampicillin and isolated using standard techniques (Sambrook et al.,1989). The plasmid DNA was checked for insert by appropriate restrictionnucleases (e.g. EcoRI, NcoI, XbaI) and was shown by sequence analysis tocontain the proper sequence of the GLP-1 analogue precursors molecule(R34)GLP-1(9-37).

The plasmid was transformed into S. cerevisiae strain ME1719 and a yeasttransformant harbouring the plasmid was selected as described inexample 1. The resulting yeast strain expressing (R34)GLP-1(9-37) wasnamed γSA96.

The yeast strain was cultivated in growth media and (R34)GLP-1(9-37) wasrecovered from the media as described in example 1.

Example 3 Construction of Yeast Expression Systems for GLP-1 Precursors

The leader and Kex2p cleavage sites were optimized in order to augmentexpression yield of processed, secreted peptide. Synthetic DNA fragmentsencoding various leader and (R34)GLP-1(11-37), or (R34)GLP-1(9-37)constructs were amplified by standard PCR. The forward primers includedan NcoI-site and the sequence encoding the optimized leader sequence.The reverse primers included an XbaI-site. Hence, allowing cloning ofthe NcoI-XbaI restricted PCR-fragments into the NcoI-XbaI restrictedcPOT-type expression vector. Refer to FIG. 4 for sequences, plasmidnumbering of (R34)GLP-1(11-37) encoding expression construct and strainnames.

Expression plasmids were propagated in E. coli in the presence ofampicillin and isolated using standard techniques (Sambrook et al.,1989). Plasmid constructs were checked by restriction endonucleasedigestion using appropriate restriction enzymes. Encoding sequences weresequence verified.

Plasmids were transformed into the S. cerevisae strain ME1719. Yeastcells harboring the plasmid were selected as described in example 1.Cultivation of the yeast strains was essentially as described in example1

Optimization of the sequence in the vicinity of the Kex2p cleavage-site,P1 to P6, led to a modest increase in yield (FIG. 4: B). Incorporationof a Proline in the sequence (P3 to P6) was explored as a potentialmeans of increasing exposure of the Kex2p cleavage site to Kex2p.Although, this turned out to not to be the case for (R34)GLP-1(11-37) abeneficial effect may be observed for (R34)GLP-1(11-37) (FIG. 4:H, I).Variation of residues P7 to P11 had a pronounced effect on expressionyield, 2-2.5 fold increases were observed when motifs containing chargedamino acids were introduced (FIG. 4: M-Z, K5-7, L2-4). A series ofconstructs had been designed for processing with DAP-1 (dipeptidylaminopeptidase) in the downstream process (FIG. 4: G, J, K, L).Constructs J and L contain a Q in the position before Thr-11, to protectagainst promiscuous cleavage by DAP-1 of the GLP-1 peptide. This Gln cansubsequently be removed using the enzymes Q-cyclase and pGAPase(Qiagen).

TABLE 1 STRAIN SEQUENCE SEQUENCE REF. YIELD PLASMID NAME RARYKR (A) 25pISNN546 SCI50 RDLGKR (B) 125 pISNN547 SCI51 RDLAKR (C) 88 pISNN548SCI52 RARAKR (D) 58 pISNN549 SCI53 RALDKR (E) 58 pISNN550 SCI54 RALAKR(F) 43 pISNN551 SCI55 PRDLGKR (H) 68 pISNN585 SCI57 RPLGKR (I) 58pISNN586 SCI58 RDLGKREA (G) 200 pISNN589 SCI56 RDLGKREAQ (J) 188pISNN599 SCI59 RDLGKREAEA (K) 240 pISNN590 SCI60 RDLGKREALEKR (K5) n.d.pISNN654 RDLGRREALEKR (K6) n.d. pISNN655 RDLGEALEKR (K7) 100 pISNN652RDLGKREAEAQ (L) 253 pISNN587 SCI44 RDLGKREAEAQKR (L2) 25 pISNN650RDLGRREAEAQKR (L3) n.d. pISNN656 RDLGEAEAQKR (L4) 13 pISNN651 ERLERDLGKR(M) 225 pISNN610 SCI63 KERLERDLGKR (N) 100 pISNN611 SCI64 ERLEKR (O) 138pISNN617 SCI65 KERLEKR (P) 125 pISNN612 SCI66 PERLERDLGKR (Q) 73pISNN613 SCI67 PERLEKR (R) 125 pISNN614 SCI68 EAEARDLGKR (S) 175pISNN615 SCI69 PEAEARDLGKR (T) 100 pISNN616 SCI70 EEAEKR (U) 125pISNN623 SCI71 EEAERDLGKR (V) 218 pISNN624 SCI72 RDLGEEAEKR (X) 218pISNN625 SCI73 EEAELAKR (Y) 165 pISNN626 SCI74 EEAELGKR (Z) 105 pISNN627SCI75 KR 100 pISNN545 SCI43

Example 4 Preparation of N-Terminal ExtensionBoc-His(Boc)-Aib-Glu(O-tBu)-Gly-OH

2-chlorotritylchloride resin (1% DVB, 1.4 mmol/g, 10 g, 14.0 mmol) waspre-swelled in DCM before it was added a solution of Fmoc-Gly-OH (8.3 g,28 mmol) and DIPEA (9.03 g, 70 mmol) in 100 mL DCM, and agitated for 30min at r.t. The resin was washed with NMP (2×100 mL) and 2×100 mLDCM-MeOH-DIPEA (80:15:5, 10 min) then NMP (3×100 mL), before treatmentwith 20% piperidine in NMP (3×100 mL) for 10 min each. The resin waswashed with NMP (6×100 mL), DCM (2×100 mL) and MeOH (2×100 mL) and driedover night in vacuo. The resin was treated with 100 mL 20% piperidine inNMP for 10 min. The step was repeated twice before it was washed withNMP (5×100 mL).

Fmoc-Glu(O-tBu)-OH (23.8 g, 56 mmol) was dissolved in a mixture of 100mL NMP and 20 mL DCM. HOAt (7.62 g, 56 mmol) was added followed by dropwise addition of DIC (7.07 g, 56 mmol). The mixture was stirred for 15min before DIPEA (9.03 g, 70 mmol) was added, and the mixture wastransferred to the resin. The resin was agitated for 16 h before it waswashed with NMP (4×100 mL). The resin was treated with 20% piperidine inNMP (100 mL) for 10 min. The step was repeated twice before it waswashed with NMP (5×100 mL).

Fmoc-Aib-OH (18.22 g, 56 mmol) was dissolved in a mixture of 100 mL NMPand 20 mL DCM. HOAt (7.62 g, 56 mmol) was added followed by drop wiseaddition of DIC (7.07 g, 56 mmol). The mixture was stirred for 20 minbefore it was added to the resin. The mixture was agitated for 30 minbefore DIPEA was added. The mixture was agitated for 16 h before theresin was washed with NMP (4×100 mL). The resin was treated with 20%piperidine in NMP (100 mL) for 10 min. The step was repeated twicebefore it was washed with NMP (5×100 mL).

Boc-His(Boc)-OH (19.9 g, 56 mmol) was dissolved in a mixture of 100 mLNMP and 20 mL DCM. HOAt (7.62 g, 56 mmol) was added followed by dropwise addition of DIC (7.07 g, 56 mmol). The mixture was stirred for 20min before it was added to the resin. The mixture was agitated for 30min before DIPEA was added. The mixture was agitated for 16 h before theresin was washed with NMP (3×100 mL), DCM (4×100 mL).

Trifluoroethanol-DCM (1:4, 125 mL) was added and the resin was agitatedfor 1 hour. The filtrate was collected and a new portion oftrifluoroethanol-DCM (1:4, 125 mL) was added and agitated for 15 min.The filtrate was collected and the solvent was removed in vacuo to givea clear oil. Ice cold diethyl ether (300 mL) was added whereupon a whiteprecipitate was formed. Light petrol ether (100 mL) was added and theprecipitate was filtered off, washed with diethyl ether and dried invacuo.

Yield: 7.3 g

HPLC (Method 01_B4): Rt=7.8 min

LCMS:m/z=944 (M+H)⁺

Calculated MW=943.2

Example 5 Preparation of N-Terminal ExtensionBoc-His(Boc)-Aib-Glu(O-Tbu)-Gly-OSuc

A solution of Boc-His(Boc)-Aib-Glu(O-tBu)-Gly-OH (1.0 g, 1.47 mmol) indry THF (60 mL), was added DIPEA (0.47 g, 3.66 mmol) andO—(N-succinimidyl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(1.10 g, 3.66 mmol) and the reaction mixture was stirred for 24 h atroom temperature. Dichloromethane (250 mL) was added and the solutionwas washed with H₂O (2×100 mL). The organic phase was dried over MgSO4,filtered and concentrated in vacuo to give 1.1 g ofBoc-His(Boc)-Aib-Glu(O-tBu)-Gly-OSu.

HPLC (Method 01_B4):Rt=8.3 min

LCMS:m/z=780.9 (M+H)⁺

Calculated MW=779.9

Example 6 Preparation of acylating agent17-((S)-1-tert-Butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)heptadecanoicacid tert-butyl ester

The title compound was prepared by the general method for thepreparation and activation of acylating agents A-OH A-B-C-D-OH,A-C-D-OH, A-B-C—OH as described above and similar to example 4.

HPLC (Method 01_B4):Rt=16.2 min

LCMS:m/z=944 (M+H)⁺

Calculated MW=943.2

Example 7 Preparation of acylating agent17-((S)-1-Carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]ethylcarbamoyl}propylcarbamoyl)-heptadecanoicacid

17-((S)-1-tert-Butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)ethoxy]ethylcarbamoyl}methoxy)ethoxy]-ethylcarbamoyl}propylcarbamoyl)heptadecanoicacid tert-butyl ester was dissolved in TFA and the resulting solutionwas stirred for 2 hr. and evaporated in vacuo to dryness. The resultingoil was coevaporated with toluene thrice to dryness resulting in an oilyresidue

LCMS: m/z=831 (M+H)⁺

Example 8 Preparation of GLP-1 derivative[Arg34]GLP-1-(11-37)-N-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl]-[Aib8,Arg34]GLP-1-(7-37)

[Arg34]GLP-(11-37) (50 mg, 0.017 mmol) was dissolved in 2 mL water andDIPEA (89 μL, 0.51 mmol) was added. The mixture was stirred for 10 minbefore pH was measured to 10.6. Then a solution of17-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxopyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]-ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}propylcarbamoyl)-heptadecanoicacid tert-butyl ester (20.5 mg, 0.022 mmol) in MeCN (1.5 mL) was addeddropwise over 10 min. The reaction mixture was stirred for 1 hour beforeMeCN was removed in vacuo and the solution was lyophilized.

The resulting residue was dissolved in NMP (2 mL) whereupon DIPEA (22μL, 0.17 mmol) and Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OSu (26 mg, 0.033mmol) was added. The reaction mixture was stirred for 4 hours before anadditional portion of His(Boc)-Aib-Glu(OtBu)-Gly-OSu (13 mg 0.017 mmol)was added and the reaction mixture was stirred over night. Ice colddiethyl ether (20 mL) was added whereupon a white precipitate wasformed. The precipitate was isolated by centrifugation and thesupernatant was decanted off. The precipitate was washed with 10 mLdiethyl ether and dried in the air.

Deprotection

The crude intermediate was dissolved in TFA-triisopropylsilane-H₂O(95:2.5:2.5, mL) and stirred for 3 h whereupon the solution wasconcentrated in vacuo to app. 1 mL. Ice cold diethyl ether (20 mL) wasadded whereupon a precipitate was formed. The precipitate was isolatedby centrifugation and the supernatant was decanted off. The precipitatewas washed with diethyl ether and dried. The crude compound wasdissolved in acetic acid-H₂O (3:7) (10 mL) and purified on HPLC.

HPLC (Method 02_B4): Rt=9.6 min

LCMS:m/z=1372.5 (M+3H)³⁺

Calculated MW=4113.7

Example 9 Preparation of GLP-1 derivativeN-epsilon26-[2-(2-[2-(2-[2-(2-[4-(17-tert-butoxycarbonylheptadecanoyl-amino)-4(S)-tert-butoxycarbonylbutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]-[Arg34]GLP-1-(9-37)peptide

To a solution of17-{(S)-1-tert-butoxycarbonyl-3-[2-(2-{[2-(2-carboxymethoxyethoxy)ethylcarbamoyl]methoxy}ethoxy)ethylcarbamoyl]propylcarbamoyl}heptadecanoic acid tert-butyl ester (94.0 mg, 0.112 mmol) inNMP (2.0 ml) was added O-(1-succinimidyl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (TSTU, 41.7 mg, 0.139 mmol) and TEA (96.0 μl, 0.69mmol). After stirring for 30 min. at room temperature the mixture wasadded drop wise to a solution of [Arg34]GLP-1-(9-37) peptide (220 mg,0.069 mmol) in water (9 ml) and TEA (96.0 μl, 0.69 mmol). After stirringfor additional 30 min at room temperature the mixture was diluted withwater (30 ml) and lyophilized for 16 h giving an oil/amorphous mixturewhich was dissolved in a mixture of acetic acid-acetonitrile-water(30:20:50, 180 ml), filtered and purified by HPLC to giveN-epsilon26-[2-(2-[2-(2-[2-(2-[4-(17-tert-butoxycarbonylheptadecanoyl-amino)-4(S)-tert-butoxycarbonylbutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]-[Arg34]GLP-1-(9-37)peptide.

HPLC (Method 02_B4_(—)4): Rt=12.1 min

LCMS:m/z=1335.5 (M+3H)³⁺

Calculated MW=4003.6

Example 10 Preparation of GLP-1 analogueN-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]ethoxy}ethoxy)acetylamino]-ethoxy}ethoxy)-acetyl][Aib8,Arg34]GLP-1-(7-37)peptide

To a solution of Boc-His(Boc)-Aib-OH (23 mg, 0.052 mmol) in NMP (2.0 ml)were added 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 20 mg, 0.052 mmol) and TEA (29 μl, 0.208mmol). After stirring for 45 min. the mixture was added to a solution ofN-epsilon26-[2-(2-[2-(2-[2-(2-[4-(17-tert-butoxycarbonylheptadecanoylamino)-4(S)-tert-butoxycarbonylbutyrylamino]ethoxy)ethoxy]acetylamino)-ethoxy]ethoxy)acetyl]-[Arg34]GLP-1-(9-37)peptide (83 mg, 0.021 mmol) and TEA 3.6 ul, 0.026 mmol). After stirringfor 14 days the reaction mixture was diluted with water (100 ml) andpurified by HPLC. The purified fractions were pooled and the organicsolvent removed in vacuo before the residue was lyophilized for 16 h.

To the residue was added a mixture oftrifluoroacetic-triisopropylsilane-water (94:3:3, 10 ml). After stirringfor 2 hours the reaction mixture was evaporated in vacuo, the residuewas dissolved in a mixture of water-ammonia (99:1, 100 ml) and purifiedby preparative HPLC to give the title compound.

HPLC (Method 02_B4_(—)4): Rt=9.4 min

LCMS:m/z=1372 (M+3H)³⁺

Calculated MW=4113.7

Example 11 Preparation of acylating agent20-[4-((S)-1-tert-Butoxycarbonyl-3-{2-[2-({2-[2-(2,5dioxo-pyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]ethylcarbamoyl}methoxy)ethoxy]ethylcarbamoyl}-propylcarbamoyl)piperidin-1-yl]-20-oxo-icosanoicacid tert butyl ester

The title compound was prepared by the general method for thepreparation and activation of acylating agents A-OH A-B-C-D-OH,A-C-D-OH, A-B-C—OH as described above and similar to example 4.

LCMS: m/z=1111 (M+H)⁺

LCMS:m/z=1082.6 (M+H)⁺

Calculated MW=1082.4

Example 12 Preparation of GLP-1 analogue [Desamino-His7,Glu22,Arg26,34,Lys37(Lys(2-{2-[2-(2-{2-[2-((S)-4-Carboxy-4-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}-butyrylamino)ethoxy]-ethoxy}-acetylamino)ethoxy]ethoxy}acetyl)]-GLP-1(7-37) peptide

To a solution of [Glu22,Arg26,34,Lys37]GLP-1-(8-37) peptide (46 mg,0.013 mmol) in 2 mL water was added DIPEA (50.4 mg, 0.39 mmol) and themixture was stirred for 10 min before a solution of20-[4-((S)-1-tert-butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]-ethylcarbamoyl}-propylcarbamoyl)-piperidin-1-yl]-20-oxo-icosanoicacid tert butyl ester (32.4 mg, 0.03 mmol) in 1 mL MeCN was addeddropwise over the course of 15 min. The reaction mixture was stirred for20 min before water (20 mL) was added, and the solution was freeze dried

The resulting residue was dissolved in NMP (1 mL) and4-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)-ethyl]-imidazole-1-carboxylicacid adamantan-1-yl ester (Adoc-des amino His-OSu ester) mixture wasstirred for 3 hours before it was added drop wise to ice cold diethylether (20 mL). The white precipitate was isolated by centrifugation andwashed twice with diethyl ether (20 mL) and dried.

The resulting residue was treated with TFA-TIPS-water (95:2.5:2.5, 1 mL)for 3 hours before it was added drop wise to ice cold diethyl ether (10mL). The white precipitate was isolated by centrifugation and washedtwice with diethyl ether (10 mL). The residue was purified bypreparative HPLC to give the title compound

HPLC (Method 01_B4_(—)2): Rt=11.1 min

LCMS:m/z=1484 (M+3H)³⁺

Calculated MW=4452.1

Example 13 Preparation of4-[2-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)ethyl]imidazole-1-carboxylicacid adamantan-1-yl ester

4-(2-Carboxyethyl) imidazole-1-carboxylic acid adamantan-1-yl ester (1g, 3.1 mmol) was dissolved in THF (5 mL) and DMF (10 mL). The solutionwas cooled to 0° C. and TSTU and DIEA were added. The solution wasstirred at 0° C. for 1 h, and at room temperature for 16 h. The samplewas concentrated under vacuum. EtOAc (100 mL) was added, and thesolution was washed with 0.2 N HCl (2×50 mL), dried over MgSO₄, andconcentrated under vacuum to yield a sticky crystalline residue (1.18 g,90% yield). The crude product was used without further purification.

LCMS:m/z=416.2 (M+H)

¹H-NMR (DMSO, 300 MHz) (selected signals) δ 8.22 (s, 1H), 7.37 (s, 1H),3.02 (t, 2H), 2.82-2.88 (m, 2H), 2.81 (s, 4H), 2.19 (s-br, 9H), 1.66 (s,6H).

Example 14 Preparation of GLP-1 analogue N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Glu22, Arg26,34, Lys 37] GLP-1 (8-37) peptide

The GLP-1 analogue was prepared using a CEM Liberty peptide synthesizerstarting with a Fmoc-Lys(Mtt)-wang resin. After the Fmoc group from Ala8was removed, the N-terminal was protected by performing a doublecoupling with di-tert-butyl dicarbonate. The Mtt group was removed bywashing the resin with DCM, treating the resin withhexafluoroisopropanol for 15 min at r.t and washing the resin with DCM.The N-epsilon 37 modifications were prepared on the CEM Libert peptidesynthesizer using Fmoc-protected reagents and eicosanedioic acidmono-tert-butyl ester. The crude peptide was cleaved from the resinTFA-TIPS-Water (95:2.5:2.5) and precipitated in ice cold ether, andisolated by centrifugation. The crude peptide was purified bypreparative HPLC (4 cm dia.×200 mm, C18, 60 ml/min, 33-53%acetonitrile).

LCMS:m/z=1065.8 (M+4H)⁴⁺

Example 15 Preparation of GLP-1 analogue N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Desamino-His(Adoc)₇, Glu22, Arg26,34, Lys 37] GLP-1 (7-37) peptide

The GLP-1 analogue N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Glu22, Arg26,34, Lys 37] GLP-1 (8-37) peptide (2.1 mg, 0.001 mmol) wasdissolved in NMP (105 μL). A solution of4-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)ethyl]imidazole-1-carboxylicacid adamantan-1-yl ester was prepared by dissolving 25.3 mg in 1296 μLNMP, and 25 μL of this solution was added to the peptide solutionfollowed by DIEA (0.9 μL). The conversion to the product was followed byLCMS analysis. After 2 h at r.t, the ELS signal ratio of the product tothe starting peptide was observed to be 976:28 (97% conversion).

LCMS:m/z=1141.1 (M+4H)⁴⁺

Example 16 Preparation of acylating agent19-{[trans-4-((S)-1-Carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]-ethylcarbamoyl}-propylcarbamoyl)-cyclohexylmethyl]-carbamoyl}-nonadecanoicacid

19-{[trans-4-((S)-1-tert-Butoxycarbonyl-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]-ethylcarbamoyl}-propylcarbamoyl)-cyclohexylmethyl]-carbamoyl}-nonadecanoicacid tert-butyl ester (1.0 g, 0.9 mmol) was stirred in TFA (10 mL) for 1h 45 min before 100 mL ice cold diethyl ether was added. The whiteprecipitate was isolated by centrifugation and washed 3× with diethylether to give the title compound.

The precipitate was dried in vacuo for 16 hours.

LCMS:m/z=999 (M+1)⁺

Example 17 Preparation of GLP-1 analogueN-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)-butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl]-[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)Peptide

[Glu22, Arg26, Arg34, Lys37]GLP-1-(8-37) prepared via recombinanttechnique (300 mg, 0.09 mmol) dissolved in 6 mL water was added DIPEA(373 μL, 2.19 mmol) and stirred for 10 min whereupon a solution of19-{[trans-4-((S)-1-carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]-ethylcarbamoyl}-propylcarbamoyl)-cyclohexylmethyl]-carbamoyl}-nonadecanoicacid (175.7 mg, 0.18 mmol) in NMP (1.7 mL) was added dropwise. Themixture was stirred for 90 min before Na₂HPO₄x7H₂O (470.6 mg, 1.76 mmol)was added and pH was adjusted to 8.4 using 1 N HCl.

In another flask 3-(1H-Imidazol-4-yl)-propionic acid (93.2 mg, 0.53mmol), O—(N-succinimidyl)-N,N,′,N′-tetramethyluronium tetrafluoroborate(159.0 mg, 0.53 mmol) and DIPEA (180 μL, 1.06 mmol) was mixed in NMP(0.75 mL) and the solution was stirred for 1 hour before it was addeddrop wise to the reaction mixture containing the peptide. The mixturewas stirred for 14 hours whereupon piperidine (350 μL, 3.51 mmol) wasadded. The mixture was stirred for another two hours, before it wasdiluted with water to a total volume of 20 mL, and purified bypreparative HPLC, using a gradient from 33 to 53% MeCN in water.

LCMS:m/z=1475.8 (M+3H)³⁺

Example 18 Alternative preparation of GLP-1 analogueN-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)-acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)

[Arg34]GLP-1-(11-37) prepared via recombinant technique (100 mg, 0.033mmol) dissolved in 2 mL water was added DIPEA (113 μL, 0.66 mmol) andstirred for 10 min whereupon17-((S)-1-carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl-methoxy)-ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]-ethylcarbamoyl}-propyl-carbamoyl)-heptadecanoicacid (55 mg, 0.066 mmol) (Prepared in accordance with example 2, 3 and13) was added in small portions over 15 min. The mixture was stirred for60 min before Na₂HPO₄x7H2O (353.9 mg, 1.32 mmol) was added and pH wasadjusted to 8.3 using 1 N HCl. Fmoc-His-Aib-Glu-Gly-OSu (246.2 mg, 0.33mmol) was added to the reaction mixture containing the peptide and themixture was stirred for 14 hours whereupon piperidine (400 μL, 4.05mmol) was added. The mixture was stirred for another 30 min, before itwas diluted with water-MeCN (9:1) to a total volume of 40 mL, andpurified by preparative HPLC, using a gradient from 30 to 50% MeCN inwater.

LCMS:m/z=1372.5 (M+3H)³⁺

Example 19 Alternative preparation of GLP-1 analogueN-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)-acetylamino]ethoxy}ethoxy)acetyl][Aib8,Arg34]GLP-1-(7-37)peptide

Acylation: Recombinant [Arg34]GLP-1-(11-37) (99 mg; 33 μmol) wassuspended in H₂O (4 mL) in the 70 mL reaction chamber of a Metrohm 848Titrino plus titrator. The pH was adjusted to pH=11.3 controlled by theautotitrators SET program (by addition of 3.8 mL, 0.1 M NaOH (aq)). AtpH=11.3 the peptide was fully dissolved. The activated sidechain17-((S)-1-Carboxy-3-{2-[2-({2-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonylmethoxy)ethoxy]-ethylcarbamoyl}-methoxy)-ethoxy]ethylcarbamoyl}propylcarbamoyl)-heptadecanoicacid (55 mg, 66 μmol, 2 eq) was dissolved in NMP (250 μL) andtransferred to a 250 μL Hamilton syringe. By the action of an automatedsyringe pump 250 μL (2.0 eq) of this solution was added over 10 minutes.After additional 60 minutes a sample (10 μL) was taken out, diluted by90 μL MeCN—H₂O (1:1) HPLC showed a major peak of N26 acylated product(appr. 95%)+trace of unreacted peptide+trace of hydrolysed side chain.

Ligation: Tetrapeptide NHS ester Fmoc-His-Aib-Glu-Gly-OSu (123 mg, 165μmol, 5 eq) was dissolved in NMP (250 μL). The pH of the reactionmixture was adjusted to 6.9 with dilute acetic acid. Then pH wasadjusted to 7.0 by the pH-stat function on the titrator. The 250 μL ofNHS ester was added over 20 minutes. The pH was stabilized onpH=7.0±0.1.

Sample taken out after additional 30 min and HPLC showed a 1:1 ratio ofthe acylated intermediate and the expected product (with Fmoc protectionin the N-terminal position).

Another 5 eq of the tetrapeptide NHS ester was added as above. Thesolution was left stirring for 16 h. The ratio was now 2:8 of theacylated intermediate and the product.

Another 2.5 eq (Total of 12.5 eq) of the tetrapeptide NHS ester wasadded as above. The solution was left stirring for 16 h. Ratio was nowapprox. 1:9 of the acylated intermediate and the product.

Piperidine (1.6 mL) was added to the reaction mixture and stirringcontinued for 1 h.

HPLC showed full deprotection. The crude product was purified on reversephase HPLC to yield 62 mg (45%) of the wanted product (98% pure).

LCMS:m/z=1372.5 (M+3H)³⁺

Example 20 Preparation of Fmoc-His(Boc)-Aib-Glu(O-Tbu)-Gly-OH

Dens Mw Mol n W V Name [g/ml] [g/mol] Ratio [mmol] [g] ml Product:C41H52N6O11 804.905 1    98  78.9 1^(st) AA 2-chlorotritylchloride resin1% 1    98 140   DVB Fmoc-Gly-OH 297.3  2   196  58.3 DIPEA 0.76 129.3 2   196 66.7 pH 9 Subst. Fmoc-Gly-resin: 0.7 mmol/g 2^(nd) AAFmoc-Glu(OtBu)-OH 425.5  2   196  86.9 TCTU 355.5  2   196  69.7 DIPEA0.76 129.3  2   196 66.7 Fmoc-Glu(OtBu)-OH 425.5  0.5  49  21.7 Re-coupling TCTU 355.5  0.5  49  17.4 DIPEA 0.76 129.3  0.5  49 17.6 3^(th)AA Fmoc-Alb-OH 325.4  2   196  63.8 TCTU 355.5  2   196  69.7 DIPEA 0.76129.3  2   196 66.7 4^(th) AA Fmoc-His(boc)-OH 477.5  2   196  93.6 TCTU355.5  2   196  69.7 DIPEA 0.76 129.3  2   196 66.7

Loading of Glycine

The resin was pre-swelled in DCM (1 liter, 20 min) before it was added asolution of Fmoc-Gly-OH and DIPEA in 500 mL DCM, and agitated for 60min. The resin was washed with DMF (2×500 mL) and 2×500 mLDCM-MeOH-DIPEA (80:15:5, 10 min) then DMF (3×500 mL), DCM (500 mL) andleft overnight at RT.

Coupling 1

20% piperidine in DMF (500 mL) for 50 min. was used for Fmocdeprotection, then the resin was washed with DMF (8×500 mL).

Fmoc-Glu(OtBu)-OH and TCTU were dissolved in 700 mL DMF added to reactorand DIPEA was added. The mixture was stirred for 6 h. According HPLCtest free amine groups was still present; coupling was repeated using ¼of reagents. When coupling reaction was finished (no amino groupspresent), resin was washed with DMF (4×500 mL)

Coupling 2

Fmoc-Aib-OH and TCTU were dissolved in 700 mL DMF solution, put into thereactor and DIPEA was added. The mixture was stirred for 50 min. Whencoupling reaction was finished (no amino groups present), resin waswashed with DMF (4×500 mL).

Coupling 3

20% piperidine in DMF (500 mL) for 30 min was used for Fmocdeprotection, and then the resin was washed with DMF (8×500 mL).

Fmoc-His(Boc)-OH and TCTU were dissolved in 700 mL DMF, solution putinto the reactor and DIPEA was added. The mixture was stirred for 50min. When coupling reaction was finished (no amino groups present),resin was washed with DMF (3×500 mL) and DCM (2×500 mL).

1^(st) Cleavage

The 1^(st) cleavage was done using 1/7 of resin. Trifluoroethanol-DCM(1:4, 500 mL) was added and the resin was agitated for 70 min. Thefiltrate was collected and the solvent was removed in vacuum to giveoil. The oil was dissolved in ethyl acetate and left stay inrefrigerator to form crystals. Crystals were filtrated, washed usingdiethyl ether and dried on air, to give 6.1 g of product (yield 45%),purity by HPLC 99.0%.

2^(nd) Cleavage

The 2^(nd) cleavage was done using 3/7 of resin. Trifluoroethanol-DCM(1:4, 1000 mL) was added and the resin was agitated for 90 min. Thefiltrate was collected and the solvent was removed in vacuum to giveoil. The oil was dissolved in ethyl acetate and left stay inrefrigerator overnight to form crystals. Crystals were filtrated, washedby diethyl ether and dried on air, to give 33.1 g of product (yield80%), purity by HPLC 98.7%.

Yield: 6.1 g (45%)

ESI+MS m/z: 805.1 (M+H)⁺, 1610.7 (2M+H)⁺.

HPLC R_(t) (Luna 4.6×250, 5 ul, 100A; acetonitrile/buffer* 30:70 to60:40, 30 min, 1 ml/min., 220 nm): 23.31 min.; purity 99.0%

and 33.1 g (80%)

ESI+MS m/z: 805.1 (M+H)⁺, 1610.8 (2M+H)⁺.

HPLC R_(t) (Luna 4.6×250, 5 ul, 100A, acetonitrile/buffer* 30:70 to60:40, 30 min, 1 ml/min., 220 nm): 23.16 min.; purity 98.7%

*buffer: 2.71 g KH₂PO₄, 7.13 g NaH₂PO₄.2H₂O dissolved in H₂O (2 L);

Example 21 Preparation of Fmoc-his-Aib-Glu-Gly-Osuc, Tfa Salt

Fmoc-His(Boc)-Aib-Glu(O-tBu)-Gly-OH (1.25 g, 1.56 mmol) was dissolved inTHF (10 mL) and were added DIPEA (0.64 g, 3.73 mmol) and TSTU (0.56 g,1.87 mmol, 1.2 eq). The mixture was stirred over night.

LCMS: app. 95% conversion. The mixture was filtered and the solvent wasremoved in vacuo. EtOAc (100 mL) was added and the organic phase waswashed twice with cold 0.1 N HCl and dried over Na₂SO₄, filtered andevaporated to give crude Fmoc-His(Boc)-Aib-Glu(OtBu)-Gly-OSu (1.1 g,79%). The crude product was dissolved in DCM (2 mL) and added TFA (1mL). Stirred for 1 h. LCMS: 100% deprotected. After evaporation of theorganic solvent ice-cold diethylether was added to the residue and theresulting precipitate was collected by filtration and dried in a vacuumoven to give Fmoc-His-Aib-Glu-Gly-OSu (430 mg)

LCMS:m/z=746.3 (M+H)⁺

Example 22 Preparation of 3-(1-trityl-1H-imidazol-4-yl)-propionic acid2,5-dioxo-pyrrolidin-1-yl ester

3-(N-Tritylimidazol-4-yl)propionic acid (Jung et al., Bioorg. Med. Chem.Lett., 6, 2317-2336, 1998) (50 g, 0.13 mol) was dissolved in DCM (500ml), N-hydroxysuccinimide (24 g, 0.209 mol) andN,N′-dicyclohexylcarbodiimide (35 g 0.170 mol) was added, whilemaintaining the internal temperature below 30° C. with an icebath. Theresulting mixture was stirred at room temperature overnight. The mixturewas filtered and the resulting solution was concentrated in vacuo,re-dissolved in THF (350 ml) upon heating and mixed with 2-propanol (350ml) to a resulting temperature of 32° C., then cooled to 5° C. Thecrystalline product was filtered and washed with 2-propanol (150 ml) anddried in vacuum to afford the title compound (47.3 g, yield 75%).

¹H NMR (D₆-DMSO, 400 MHz): δ 7.15-6.99 (m, 9H), 6.82 (s, 1H), 6.81-6.62(m, 6H), 6.38 (s, 1H), 2.63 (t, 3H), 2.4 (m, 6H).

Example 23 Preparation of4-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)-ethyl]-1H-imidazol-1-iumtrifluoroacetate

3-(1-Trityl-1H-imidazol-4-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-ylester (40 g, 83 mmol) was dissolved in DCM (240 ml). Triisopropylsilane(42 ml) and trifluoroacetic acid (240 ml) was added and the resultingsolution was stirred at room temperature for 90 min. The solution wasevaporated in vacuo, redissolved in acetonitrile (120 ml), andtert-butylmethyl ether (290 ml) was added dropwise and the solution wascooled to 8° C. The precipitate was collected by filtration and dried invacuum to afford a white compound (24 g, 82%).

¹H NMR (D₆-DMSO, 400 MHz): δ 8.90 (s, 1H), 7.47 (s, 1H), 3.09 (t, 2H),2.79 (t, 2H), 2.79 (s, 4H).

Example 24

Alternative preparation of GLP-1 analogueN-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)-butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl]-[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)peptide

[Glu22,Arg26,Arg34,Lys37]GLP-1-(8-37) peptide (80 g, 0.023 mol) preparedvia recombinant technique was dissolved in water (6400 ml) andtriethylamine (28 ml) was added to pH 11.0.(22S)-22-{[(trans-4-{[(19-carboxynonadecanoyl)amino]methyl}-cyclohexyl)carbonyl]amino}-1-[(2,5-dioxo-1-pyrrolidinyl)oxy]-1,10,19-trioxo-3,6,12,15-tetraoxa-9,18-diazatricosan-23-oicacid (34.9 g, 0.035 mol) was dissolved in NMP (210 ml) and added to thesolution over 2 hr., while maintaining the pH between 11.0-11.1 bycontinuous addition of triehylamine. The pH was adjusted to 7.0 byaddition of 1M sulphuric acid (aq, 140 ml).4-[2-(2,5-Dioxo-pyrrolidin-1-yloxycarbonyl)-ethyl]-1H-imidazol-1-iumtrifluoroacetate (36.99 g, 0.105 mol) was dissolved in NMP and added tothe solution over 2 hr. while maintaining the pH at 7.0-7.1 bycontinuously addition of triethylamine. The pH was adjusted to 11.2 byaddition of 1 M NaOH (aq, 500 ml) and the solution was stirred at roomtemperature for 7 hr. pH was adjusted to 7.4 by addition of 1M sulphuricacid (aq, 140 ml), to a total of 7973.8 g which was purified bypreparative HPLC.

LCMS: m/z=1476 (M+3H)³⁺

Example 25 Preparation of [Aib8,Arg34]GLP-1-(7-37) peptide

Arg34-GLP-1[9-37]•4TFA (103 mg; 0.028 mmol) was dissolved in 2 mL waterand transferred to a titrator reaction chamber with additional 2×1 mLwater. The reaction chamber was equipped with a stirring magnet, pHelectrode and a titrant tube. The pH of the solution was 1.9. Thetitrator was programmed to a ‘STAT’ method at fixed pH=11.3. Thetitrant, sodium hydroxide (3.27 mL, 0.1 M aq), was added by the titratorto reach pH=11.3. (Boc)₂O (186 mg, 0.85 mmol, 3 eq) was dissolved in NMP(2.500 mL) and a 250 μL Hammilton syringe was filled with this solution(i.e. 250 μL; 18.6 mg (Boc)₂O, 3 eq).

Water (1.0 mL) was added to the reaction chamber to make a total volumeof 8.3 mL and the (Boc)₂O NMP solution was then added over 20 min by aidof a syringe pump at room temperature. The titrator was keeping thepH=11.3 by automatic addition of the titrant. After stirring foradditional 10 minutes LCMS showed 88% of the Lys26(NHBoc) producttogether with a diboc product (9%) and starting material (3%). The pH ofthe solution was adjusted to 6.5 with a small volume of dilute HOAc (aq)and the titrator was set at pH=7.5 for the further reaction.

A solution of Boc-His(Boc)-Aib-OSu (305 mg, 0.568 mmol, 20 eq) wasdissolved in NMP (500 μL) giving a total volume of 715 μL. Of thissolution 2×250 μL (2×7 eq) was added to the reaction mixture over 2×30minutes keeping pH=7.5. Immediate precipitation of theBoc-His(Boc)-Aib-OSu was observed, but upon stirring all dissolved.After end of addition the mixture was stirred for another 2 h at pH=7.5and at room temperature to allow dissolution and reaction of thereactant.

The mixture was lyophilized and treated with TFA containing 3% TIPS and3% water for 2 h. The solvents were evaporated and the residue waspurified by HPLC to give the desired product.

LCMS m/z: 849.88 (M+4H)⁴⁺, 1132.86 (M+3H)³⁺. Calcd. 3396.698 (M+H)⁺.

Example 26 Preparation of Boc-His(Boc)-Aib-OH

Loading of Aib: 2-Chlortritylchloride resin 1% DVB (Load 1.1 mmol/g)(10.0 g, 11 mmol) was pre-swelled in DCM before it was added a solutionof Fmoc-Aib-OH (7.16 g, 22 mmol) and DIPEA (9.4 mL, 55 mmol) in DCM (100mL). The mixture was agitated for 60 min. The resin was washed with NMP(2×100 mL) and DCM-MeOH-DIPEA (80:15:5) (2×100 mL, 10 minutes each), andNMP (3×100 mL). The washed resin was treated with 20% piperidine in NMP(3×100 mL, 10 min each). The resin was washed with NMP (6×100 mL).

Coupling: Boc-His(Boc)-OH (15.64 g, 44 mmol) was dissolved in NMP (100mL) and DCM (20 mL) and HOBt (5.95 g, 44 mmol) was added followed byslow addition of DIC (6.81 mL, 44 mmol). The mixture was stirred for 15min before DIPEA (9.40 mL, 55 mmol) was added. The activated mixture wasadded to the resin and the resin was agitated for 16 h before it waswashed with NMP (4×100 mL) and DCM (10×100 mL).

Cleavage: Trifluoroethanol-DCM (1:4, 100 mL) was added and the resin wasagitated for 1 hour. The filtrate was collected and a new portion oftrifluoroethanol-DCM (1:4, 100 mL) was added and agitated for 15 min andthe filtrate was collected. The combined filtrates were evaporated invacuo and petrolether (50 mL) was added. The resulting precipitate waswashed with petrolether, collected and dried in vacuum oven for 16 h at40° C. to give Boc-His(Boc)-Aib-OH (1.00 g, yield 21%, purity 95%).

LCMS m/z: 441.18 (M+H)⁺. Calcd. 440.227 (M+H)⁺.

Example 27 Preparation of Boc-His(Boc)-Aib-Osu

Boc-His(Boc)-Aib-OH (0.70 g, 1.59 mmol) was dissolved in THF (5.0 mL)and TSTU (1.44 g, 4.77 mmol) and DIPEA (1.36 mL, 7.95 mmol) was added.The reaction mixture was stirred at rt. for 2 h. LCMS showed almostcomplete conversion into the oxazolone product. The reaction mixture wasfiltered and the solvent was evaporated. The residue was re-dissolved inDCM (10 mL) and N-hydroxysuccinimide (292 mg, 2.56 mmol, 1.6 eq) wasadded. The mixture was stirred over night at room temperature. HPLC showa major peak of the desired Su-ester (85%), trace of un-reacted startingmaterial and 5% of the ring-closed oxazolone. The solvent was evaporatedat room temperature under reduced pressure and H₂O (25 mL) was added todissolve unreacted N-hydroxysuccinimide. The H₂O was decanted off andthe residue was dissolved in EtOAc (100 mL) dried over MgSO₄, filteredand evaporated to give a white foam containing 70% of the desiredproduct.

LCMS m/z: 538.18 (M+H)⁺. Calcd. 538.251 (M+H)⁺

Example 28 Preparation of Fmoc-his-Aib-Osu

Fmoc-His(Trt)-Aib-OH (5 g, 7.1 mmol) was dissolved in THF (50 ml). DCC(1.61 g, 7.8 mmol) and NHS (0.94 g, 8.2 mmol) was added and theresulting solution was stirred for 16 hr at room temperature. The DCUwas filtered and the solution was evaporated in vacuo. The resulting oilwas dissolved in DCM (34 ml) and trifluoroacetic acid (34 ml) and TIPS(6 ml) was added. The solution was stirred for 2 hr at room temperature,evaporated in vacuo to 25 ml, added dropwise to TBME (200 ml), stirredfor 60 min, and the resulting white percipetate is collected byfiltration, and washed with additional TBME. The collected precipitatewas dried in vacuum for 16 hr.

LCMS: m/z=560 (M+H)⁺

Example 29 Preparation of GLP-1 derivative [Aib8,Arg34]GLP-1-(7-37)Peptide

[Arg34] GLP-1(9-37) peptide•4TFA (103 mg; 0.028 mmol) was dissolved in0.1 M TEA(aq., 7.5 ml) additional TEA was added (140 μl) to a resultingpH of 11.28. FmocOSu (90 mg) was dissolved in NMP (1600 μl) and 334 μl(0.055 mmol) of this solution was added dropwise to the aqueous peptidesolution. pH was adjusted to 7 by addition of 1M H₂SO₄(aq).FmocHisAibOSu trifluoroacetate salt (90 mg, 0.134 mmol) was dissolved inNMP (800 μl) and added dropwise to the solution while maintaining the pHbetween 7.0 and 7.5 by continuos addition of 1M NaOH(aq). Piperidin (2ml) was added and the resulting solution was stirred for 60 min. at roomtemperature. TBME (5 ml) was added and the mixture was stirred for 30min. The aqueous phase was separated and lyophilised.

LCMS: m/z=1134 (M+3H)³⁺

1. A method for making a GLP-1 analogue or derivative comprising one ormore non-proteogenic amino acids in the N-terminal part, said methodcomprising the steps of: i) culturing a host cell comprising anucleotide sequence encoding a precursor molecule of said GLP-1 analogueunder suitable conditions for expression of said precursor molecule, ii)separating the expressed precursor molecule from the culture broth, iii)coupling an N-terminal amino acid extension comprising one or morenon-proteogenic amino acids to the expressed precursor molecule, iv)isolating the resulting GLP-1 analogue or derivative by suitable means.2. A method according to claim 1 wherein the lysine group(s) of theprecursor molecule are protected after step ii) and deprotected againafter coupling of the N-terminal extension.
 3. A method according toclaim 1 wherein the N-terminal amino acid extension is protected beforeits use in step iii) and deprotected again after coupling of theN-terminal extension.
 4. A method according to claim 1, wherein theprecursor molecule of said GLP-1 analogue is selected from the list ofprecursor molecules comprising the amino acid sequence of the generalformula: Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄-Xaa₄₅

wherein Xaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr orGln; Xaa₂₀ is Leu or Met; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys orArg; Xaa₂₅ is Ala or Val; Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu;Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu,Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆ is Arg, Gly or Lys; Xaa₃₇ is Gly,Ala, Glu, Pro, Lys, or is absent; Xaa₃₈ is Lys, Ser, or is absent; Xaa₃₉is Ser, Lys, or is absent; Xaa₄₀ is Gly, or is absent; Xaa₄₁ is Ala, oris absent; Xaa₄₂ is Pro, or is absent; Xaa₄₃ is Pro, or is absent; Xaa₄₄is Pro, or is absent; Xaa₄₅ is Ser, or is absent; provided that ifXaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ or Xaa₄₅ is absent theneach amino acid residue downstream is also absent.
 5. A method accordingto claim 1, wherein the precursor molecule of said GLP-1 analogue isselected from the list of precursor molecules comprising the amino acidsequence of the general formula:Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃-Xaa₄₄- Xaa₄₅

wherein Xaa₉ is Glu or Asp Xaa₁₀ is Gly or Ala Xaa₁₆ is Val or Leu;Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr or Gln; Xaa₂₀ is Leu or Met;Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys or Arg; Xaa₂₅ is Ala or Val;Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu; Xaa₃₀ is Ala, Glu or Arg;Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu, Asn, His or Arg; Xaa₃₅ isGly; Xaa₃₆ is Arg, Gly or Lys; Xaa₃₇ is Gly, Ala, Glu, Pro, Lys, or isabsent; Xaa₃₈ is Lys, Ser, or is absent; Xaa₃₉ is Ser, Lys, or isabsent; Xaa₄₀ is Gly, or is absent; Xaa₄₁ is Ala, or is absent; Xaa₄₂ isPro, or is absent; Xaa₄₃ is Pro, or is absent; Xaa₄₄ is Pro, or isabsent; Xaa₄₅ is Ser, or is absent; provided that if Xaa₃₈, Xaa₃₉,Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ or Xaa₄₅ is absent then each aminoacid residue downstream is also absent.
 6. A method according to claim1, wherein the precursor molecule of said GLP-1 analogue is selectedfrom the list of precursor molecules comprising the amino acid sequenceof the general formula: Xaa₈-Xaa₉-Xaa₁₀-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Xaa₂₃-Ala-Xaa₂₅-Xaa₂₆-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Xaa₄₂-Xaa₄₃- Xaa₄₄-Xaa₄₅

wherein Xaa₈ is Ala, Gly, Val, Leu, Ile, Lys Xaa₉ is Glu, Asp Xaa₁₀ isGly, Ala Xaa₁₆ is Val or Leu; Xaa₁₈ is Ser, Lys or Arg; Xaa₁₉ is Tyr orGln; Xaa₂₀ is Leu or Met; Xaa₂₂ is Gly or Glu; Xaa₂₃ is Gln, Glu, Lys orArg; Xaa₂₅ is Ala or Val; Xaa₂₆ is Lys, Glu or Arg; Xaa₂₇ is Glu or Leu;Xaa₃₀ is Ala, Glu or Arg; Xaa₃₃ is Val, Lys or Arg; Xaa₃₄ is Lys, Glu,Asn, His or Arg; Xaa₃₅ is Gly; Xaa₃₆ is Arg, Gly or Lys; Xaa₃₇ is Gly,Ala, Glu, Pro, Lys, or is absent; Xaa₃₈ is Lys, Ser, or is absent; Xaa₃₉is Ser, Lys, or is absent; Xaa₄₀ is Gly, or is absent; Xaa₄₁ is Ala, oris absent; Xaa₄₂ is Pro, or is absent; Xaa₄₃ is Pro, or is absent; Xaa₄₄is Pro, or is absent; Xaa₄₅ is Ser, or is absent; provided that ifXaa₃₈, Xaa₃₉, Xaa₄₀, Xaa₄₁, Xaa₄₂, Xaa₄₃, Xaa₄₄ or Xaa₄₅ is absent theneach amino acid residue downstream is also absent.
 7. A method accordingto claim 1, wherein the non-proteogenic amino acids in the N-terminalamino acid extension comprising one or more non-proteogenic amino acids,are selected from the group consisting of γ-carboxyglutamate, ornithine,phosphoserine, D-amino acids such as D-alanine and D-glutamine,D-alanine and D-leucine, Aib (α-aminoisobutyric acid), Abu(α-aminobutyric acid), Tle (tert-butylglycine), 3-aminomethyl benzoicacid, anthranilic acid, des-amino-Histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, Nα-acetyl-histidine, α-fluoromethyl-histidine,α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine,4-pyridylalanine, (1-aminocyclopropyl) carboxylic acid,(1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl) carboxylicacid, (1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl)carboxylic acid, (1-aminocyclooctyl) carboxylic acid, α-methyl prolin,1-methyl histidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine.
 8. A method according to claim 1,wherein the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids has the general formula Xaa₇-Xaa₈-Xaa₉-Xaa₁₀

wherein Xaa₇ is selected from L-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, α-fluoromethyl-histidine,α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine and4-pyridylalanine, 1-methyl histidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine Xaa₈ is selected from Ala, Gly, Val, Leu,Ile, Lys, Aib, (1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl) carboxylic acid,(1-aminocyclohexyl) carboxylic acid, (1-aminocycloheptyl) carboxylicacid, or (1-aminocyclooctyl) carboxylic acid and alpha-methyl prolineXaa₉ is selected from Glu, Asp, γ,γ-dimethyl Glu, β,β-dimethyl Glu andβ,β-dimethyl Asp, and Xaa₁₀ is selected from Gly, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, and(1-aminocyclooctyl) carboxylic acid.
 9. A method according to claim 1,wherein the N-terminal amino acid extension comprising one or morenon-proteogenic amino acids has the general formula Xaa₇-Xaa₈

wherein Xaa₇ is selected from L-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, N^(α)-acetyl-histidine, α-fluoromethyl-histidine,α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine and4-pyridylalanine; 1-methyl histidine, 3-methyl histidine,4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine; and Xaa₈ is selected from Gly, Aib,(1-aminocyclopropyl) carboxylic acid, (1-aminocyclobutyl) carboxylicacid, (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or(1-aminocyclooctyl) carboxylic acid and alpha-methyl proline
 10. Amethod according to claim 1, wherein the N-terminal amino acid extensioncomprising one non-proteogenic amino acid with the general formula Xaa₇wherein Xaa₇ is selected from D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine and 4-pyridylalanine; 1-methylhistidine, 3-methyl histidine, and4,5,6,7-tetrahydro-1H-imidazo[4,5-C]pyridine-6-carboxylic acidβ-(1,2,4-triazol-1-yl)-alanine.
 11. A method according to claim 10,wherein the N-terminal amino acid extension is desamino-histidine andthe coupling reaction is carried out by a4-[2-(2,5-dioxo-pyrrolidin-1-yloxycarbonyl)-ethyl]-1H-imidazol-1-iumsalt, e.g. the salt trifluoroacetate, with the expressed precursormolecule.
 12. A method according to claim 1, further comprising the stepof acylating the epsilon-amino-group of at least one lysine residue inthe expressed precursor molecule with an acylating agent, which isoptionally activated and/or protected with one or more protectiongroup(s).
 13. A method according to claim 12, wherein the acylatingagent is a carboxylic acid analogue of the general formula: A-OH,A-C-D-OH, A-B-C—OH or A-B-C-D-OH which is optionally activated and/orprotected with one or more protection group(s), wherein, A is selectedfrom the group consisting of

wherein n is selected from the group consisting of 14, 15, 16 17, 18 and19, p is selected from the group consisting of 10, 11, 12, 13 and 14,and d is selected from the group consisting of 0, 1, 2, 3, 4 and 5, andm is selected from the group consisting of 11, 12, 13, 14, 15, 16, 17, kis selected from the group consisting of 0, 1, 2, 3, 4, 5, 11 and 27,and m is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6,and R1 is a generally accepted protection group, B is selected from thegroup consisting of

wherein x is selected from the group consisting of 0, 1, 2, 3 and 4, andy is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 and 12, C is selected from the group consisting of

wherein b and e are each independently selected from the groupconsisting of 0, 1 and 2, and c and f are each independently selectedfrom the group consisting of 0, 1 and 2 with the proviso that b is 1 or2 when c is 0, or b is 0 when c is 1 or 2, and e is 1 or 2 when f is 0,or e is 0 when f is 1 or 2, wherein, when one or more free carboxylicacid(s) are present, they are optionally protected with a generallyaccepted protection group and D is selected from the group consisting of

and wherein k is selected from the group consisting of 0, 1, 2, 3, 4, 5,11 and 27, and m is selected from the group consisting of 0, 1, 2, 3, 4,5 and
 6. 14. A method according to claim 13, wherein R1 is selected fromthe group consisting of 9H-fluoren-9-ylmethoxycarbonyl (Fmoc),tert-butoxycarbonyl (Boc), Benzylcarbamate (Cbz).
 15. A method accordingto claim 1, wherein the host cell is selected from a mammalian hostcell, an avian host cell, an insect host cell, a plant host cell, abacterial host cell, a fungal host cell and a yeast host cell.